Methods of producing antibodies against morphine-heroine

ABSTRACT

Provided are bivalent immunogenic composition against morphine-heroin addiction comprising a carrier protein (“CP”) and a morphinic product, wherein the CP and the morphinic product are connected by a spacer-linker arm having a total molecular size of 16-21 Å, and methods of using the same.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/632,085, filed on Jan. 17, 2008, which claims the benefit ofand which is a national stage filing of International Application SerialNo. PCT/MX2005/000049, filed on Jul. 5, 2005, which claims priority to,and the benefit of, Mexican Patent Application Serial No.PA/A/2004/006617, filed on Jul. 7, 2004, the entire contents of both ofwhich are hereby incorporated by reference.

The present invention received support and scientific advice from Dr.Gerardo Heinze Martin and Dr. Ramón de la Fuente Muñiz. Work funded bythe Fundacion Gonzalo del Rio Arronte and the Instituto Nacional dePsiquiatría Ramón de la Fuente Muñiz (Grant 2040).

TECHNICAL FIELD

The present invention discloses a process for the preparation and use ofa bivalent vaccine against morphine-heroin addiction, which is capableto induce a robust humoral immune response against these two addictiveopiate drugs through the active immunization in mammals including thehuman. The process for the preparation of such bivalent vaccine consistsin its design, synthesis, purification, application and therapeuticvalidation. The structural formulation of this vaccine consists of theinitial synthesis and haptenization of a morphine-6-hemisuccinateintermediate derivative to the tetanus toxoid used as carrier protein.This latter chemical step is carried out using a long spacer linker armsequentially synthesized from the covalent condensation of thehomobifunctional cross-linker reagent, the1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and theheterobifunctional cross-linker reagentN-(ε-trifluoracetylcaproyloxy)-succinimide-ester (TFCS). The humoralimmune response induced by active immunization with such vaccine wascharacterized by the presence of very high and sustained titers ofcirculating polyclonal antibodies that recognize and bind withequivalent specificity both morphine and heroin in the blood, therebypreventing its blood-brain barrier permeation into the brain. Thealtered pharmacokinetics of these two drugs leads to a significantreduction of the “free” unbound fraction of morphine and heroin inplasma, thereby blunting drug entry into the brain. Thus, the antibodyantagonism on the brain barrier permeation of opiates enhances animmunoprotective mechanism that blunts the drug-reinforcing effects ofthese two opiate substances acting on the mesocorticolimbic rewardpathway, in actively vaccinated rodents with this immunogen previouslytrained to self-administered these two pharmacological reinforcers.Therefore, the present invention describes the process for thepreparation of a bivalent vaccine against morphine/heroin addictionwhich represents a new immunoreagent or pharmaceutical composition ortherapeutic formulation that can be applied, evaluated and validated asa new anti-addictive immunopharmacological therapy against these twoopiate drugs in active vaccination protocols in humans.

BACKGROUND OF THE INVENTION

The abuse of illegal substances with reinforcing addictive propertiesrepresents a major public health problem worldwide. For instance, in theUnited States of America, nearly 48 million people have been exposed toillegal drugs over a one-year period (Neurobiological Adaptations toPsychostimulants and opiates as basis of treatment development. In: NewMedications for Drug Abuse, K. Severino, A. Olivito and T. Kosten,2000). Thus, this health problem has serious and progressive deleteriouseffects on social, economic and medical areas in affected countries.Epidemiologically, the pyschostimulants such as cocaine andamphetamines, and to a lesser extent, opiate substances, like heroin andmorphine, represent the most prevalent drugs causing the highestaddictive morbidity worldwide. In developing countries like Mexico, theepidemiological data from the latest National Survey of Addictions (M.E. Medina-Mora and E. Rojas Guiot, Salud Mental, 26(2): 1-11, 2003)reported an alarming increase in the drug-intake of such substances inthe central part of the country as well in cities located between Mexicoand US border. At the clinical level, there are several co-morbidpathologies related to the addictive abuse of illegal substances, whichfall into different categories. Firstly, the high death index related tothe toxic effects induced by the overdose of such substances. Secondly,the induction of teratogenic effects in the newborn, which arefrequently associated to the chronic abuse of illegal substances byaddicted pregnant mothers. Finally, the high incidence of co-morbiddiseases of acquiring viral infections such as the humanimmunodeficiency virus (HIV), frequently detected in heroin abusers, aswell as the increased rates of crimes, violence and delinquencyfrequently associated to the drug-trade and drug-intake of such illegalsubstances. Thus, at the therapeutical level, there exist an urgent needto refocus and establish straightforward government strategies, healthprograms and novel medications to fight efficiently against drug abuseto illegal substances.

The neurobiology of drug addiction began more than three decades ago andmost of investigations have dealt with drugs' pharmacokinetics andpharmacodymamics. At the pharmacokinetic level, illegal substances ofabuse such as cocaine, morphine and heroin, exhibit potentdrug-reinforcing properties and specific pharmacokinetic profiles, whichultimately lead to their high addictive drug effects in the brain.Morphine is an alcaloid with a phenantrenic chemical structure (seeexample 1) obtained from the milky extract (opium gum) of the Papaversomniferum, and represents the main compound extracted (≧10%) togetherwith other structurally-related compounds such as codeine, tebaine, andpapaverine (C. P. O'Brien, Drug Abuse, In: The Pharmacological Basis ofTherapeutics. Pp. 621-642, 10^(th) ed. J. G. Hardman and L. E. Limbird,eds. McGraw Hill, New York, 2001). Morphine possesses a hydroxyl groupin the third position and an alcoholic hydroxyl group in the sixthposition placed within the phenolic ring structure. Conversely, heroin,a semi-synthetic derivative of the morphine, has two acetyl groupscondensed in the aforementioned positions within the opiate phenantrenicring structure (C. P. O'Brien, Drug Abuse, In: The Pharmacological Basisof Therapeutics. pp. 621.642, 10^(th) ed. J. G. Hardman and L. E.Limbird, eds. McGraw Hill, New York, 2001). Both morphine and heroin areabsorbed from the gastrointestinal and respiratory tract, including oralmucosa, as well as from the subcutaneous, intramuscular, intravascularand intrathecal spaces. These two opiate compounds display a strikingsimilar pharmacokinetic profile, based on their high blood-brain barrierpermeation capability, mostly due to their high lipophilic properties(C. P. O'Brien, Drug Abuse, In: The Pharmacological Basis ofTherapeutics. pp. 621-642, 10^(th) ed. J. G. Hardman and L. E. Limbird,eds. McGraw Hill, New York, 2001). In fact, heroin is relatively morelipophilic than morphine and thus permeates faster the blood-brainbarrier than morphine. The main catabolic route of morphine mainlyoccurs in the liver and depends on enzymatic-dependent conjugation withglucuronic acid at both the three and six hydroxyl groups placed at thephenantrenic ring structure, producing endogenous metabolites compounds,such as morphine 3-, morphine 6-, and to a lesser extent, morphine3-6-glucuronide. These catabolic intermediate compounds, represent thestructural secretory and/or excretory forms of morphine in the urine.Moreover, morphine-6-glucuronide has been shown to display a potentanalgesic and psychotropic, drug-reinforcing effects in the brain. Thus,morphine metabolites generated from the liver into the bloodstream,rapidly permeate the blood-brain barrier and activate the mu opioidreceptor subtype in the brain reward pathways mediating the reinforcingeffects of drug of abuse (L. M. Kamendulis et al., J. Pharmacol. Exper.Ther. 279:713-717, 1996; C. W. Hutto Jr. y W. Crowder, Pharmacol.Biochem. Behav. 58(1):133-140, 1997; A. J. Halliday et al., Life Sci.65(2)225-236, 1999 and D. E. Selley et al., Biochem. Pharmacol.62:447-455, 2001). In fact, recent pharmacokinetic studies (see reviewand references therein in J. Halliday et al., Life Sci. 65(2)225-236,1999) support the idea that the analgesic and/or addictive actions ofmorphine in CNS are not directly and predominantly mediated by morphineitself, but largely exerted by its glucuronated active metabolites suchas the morphine-6-glucuronide. So far, several studies (L. M. Kamenduliset al., J. Pharmacol. Exper. Ther. 279:713-717, 1996 and A. J. Hallidayet al., Life Sci. 65(2)225-236, 1999) have shown similar pharmacokineticand pharmacodynamic mechanisms for heroin. Thus, once heroin isadministered, a large fraction of the drug is rapidly catabolized in theplasma and/or liver into 6-monoacetyl-morphine, and subsequentlycatabolized into morphine and finally converted intomorphine-6-glucuronide, before reaching their neuronal targets (e.g., muopioid receptor) (R. E. Aderjan and G. Skopp, Ther. Drug Monit., 20(5):561-9, 1998). These findings support the current concept that thepharmacological agonism of heroin and its endogenous metabolites (e.g.,6-monoacetyl-morphine and morphine) on the mu opioid receptor includingthe final biotransformation active metabolites (e.g.,morphine-6-glucuronide) represents the pharmacodymamic mechanism bywhich these substances enhance their reinforcing addictive actions inthe brain (a. J. Halliday et al., Life Sci. 65(2): 225-236, 1999, D. E.Selley et al., Biochem. Pharmacol. 62:447-455, 2001 and C. P. O'Brien,Drug Abuse, In: The Pharmacological Basis of Therapeutics. pp. 621-642,10^(th) ed. J. G. Hardman and L. E. Limbird, eds. McGraw Hill, New York,2001).

Several pharmacodymic studies (see reviewed works in E. J. Nestler, Nat.Neuroci. 5:1076-1079, 2002 and P. N. Deslandes et al., J. Pharmacy andPharmacol. 54:885-895, 2002) have shown that chronic abuse to bothheroin and morphine leads to the development and establishment ofspecific long-term changes at the cellular and molecular level thatultimately produces the expression of biological neuroadaptations toopiate addiction. Moreover, these neuronal changes produce importantelectrophysiological, neurochemical and genomic changes, which areprogressively established and consolidated upon a long-term period(e.g., years) in the brain during drug addiction. Therefore, thebehavioral changes occurring during opiate addiction to these substanceof abuse in the individual, follow a time-course of increased complexityand intensity with regard to the drug addiction symptomology. Forinstance, the repetitive administration of heroin by an addict, producesan increase stereotyped compulsive behaviors leading to uncontrolleddrug-intake behaviors, associated with stereotyped rites ofadministration, initially accompanied by pharmacological tolerance andsubsequently by physical signs and symptoms of drug withdrawal afteracute suppression of the opiate drug (K. Severino et al., Ann. N.Y.Acad. Sci 909: 51-87, 2000). Thus, heroin-intake behavior becomes thehighest priority and necessity in the addicted individual, leading tothe reinstatement of compulsive drug-intake and drug-seeking behaviorsnormally observed during drug withdrawal or abstinence. Theneuroadaptative changes occurring during opiate addiction is primarilycaused by the pharmacological actions displayed by the repetitiveexposure of the drug over a clustered group of neurons localized indifferent areas of the brain (K. Severino et al., Ann. N.Y. Acad. Sci909: 51-87, 2000). These brain areas include the locus coeruleus,hippocampus, lateral hypothalamus, ventral-tegmental area, amygdaloidcomplex, nucleus accumbens and prefrontal cortex, which structurallycomprised the neuroanatomical substrate and neural pathways where opiatesubstances and other illegal drugs of abuse (e.g., cocaine) mainly exerttheir drug-rewarding and drug-reinforcing activities (K. Severino etal., Ann. N.Y. Acad. Sci 909: 51-87, 2000). In this context, the chronicadministration of both morphine and heroin induces the development of aseries of homeostatic cellular and molecular adaptive responses onneurons within the aforementioned brain structures impinged by the drug.Such adaptive responses involve several electrophysiological,biochemical and genomic alterations seen during drug addiction, whichaltogether, are produced to maintain and restore the pre-existingfunctional homeostasis of the implicated neural circuits and theiroperant neurons altered during drug abuse, prior to the compulsivedrug-intake behavior (K. Severino et al., Ann. N.Y. Acad. Sci 909:51-87, 2000). Once these neuroadaptations have been established, theabrupt suspension of the drug-intake behavior enhances the developmentof new series of neurobiological changes and cellular adaptations in theneurons impinged by the drug, leading to the neuropathological basisthat underlies the withdrawal syndrome during drug addiction. Thewithdrawal syndrome produced by both morphine and heroin in the addictedindividual, as opposed to the withdrawal syndrome induced by cocaine andamphetamine, is characterized by highly intense physical andpsychological alterations in the addicted individual (H. Ghodse, Drugsof abuse and dependence. In: Drugs and Addictive Behavior, a guide totreatment, Blackwell Science Ltd, ed., Oxford, UK, pp. 72-119, 1995; G.F. Koob, Ann. N.Y.: Acad. Sci. Vol. 909:185 2000 and K. Severino et al.,Ann. N.Y. Acad. Sci 909: 51-87, 2000). Clinically, the withdrawalsyndrome is characterized by four different stages developed in aprogressive or gradual time-course. During the first 1-7 hours, theaddict under abstinence develops behavioral manifestations characterizedby a compulsive craving and extreme anxiety for drug-intake. During asecond stage (after 8-15 hours), physical alterations such as intenselacrimation, extreme sweating, rinorrhea and lethargy are added to theinitial drug symptomology. Further on, after 16-24 hours upon continuingdrug withdrawal, physical signs such as mydriasis, piloerection,muscular cramps and changes in body temperature (e.g., intense cold andheat perception) in addition to diffuse algias, anorexia andirritability may appear as well. Subsequently, upon pesistent withdrawal(e.g., 2-6 days), other physical and behavioral signs may appear whichinclude insomnia, fever, motor delay, abdominal pain, vomiting anddiarrhea as well as increased abnormal breathing, including changes inpulse frequency and blood pressure. Thus, from the perspective ofsymptomology occurring in drug addiction, the duration and severity ofmorphine and heroin withdrawal depends on several pharmacokinetic andpharmacodynamic factors. Moreover, there has been reported that theseverity of opiate withdrawal syndrome depends on severalpharmacological and biological aspects, which include the daily amountof drug-intake (e.g., dose injected by the individual), the period oftime of drug use and/or abuse, in addition to the physical andpersonality status of the individual affecting drug-intake response.

Thus, given the complexity of the natural history of the morphine/heroinaddictive pathology, few currently available pharmacological treatmentshave been designed to modify the pharmacodynamic mechanisms by whichthese opiate substances produce their drug-reinforcing actions once theybind their specific receptor sites at their targeted neurons (D. M.Grilly, Opioids (narcotics) and their antagonists. In: Drugs and humanbehavior, 4^(th) ed. pp. 238-262, Allyn and Bacon, eds. USA, 2002). Inthis context, the acute opiate detoxification treatment represents theinitial and most currently used pharmacotherapeutic approach to treatclinically chronic addicts, which becomes a medical priority andemergency to relieve the individual's physical signs and symptoms ofdrug withdrawal, which are commonly associated with physiological,endocrinological and chemical disturbances induced by drug addiction.For example, mu opioid receptor partial agonists such as methadone andbuprenorphine, in combination with benzodiazepines and/or sedativeneuroleptics are commonly prescribed and administered for the acutedesintoxication treatment to opiates. As opposed to the acutedetoxification procedures used to treat opiate addiction, thesubstitution therapy using opiate substances such as methadone and/orbuprenorphine as well as opioid receptor antagonists, such as naloxoneand/or naltrexone, are not entirely recommended during opiatewithdrawal, because they exacerbate the demand of drug-intake behaviorof the parent opiate compounds that elicited or installed the formerdrug addictive state in the individual. Under normal circumstances, thetreatment and maintenance of opiate withdrawal syndrome requireshospitalization and clinical care with the support of specializedmedical personnel, which commonly results to be highly expensiveLikewise to the withdrawal syndrome, the complete morphine and/or heroindetoxification (suppression of drug-intake behavior) in addictedindividuals is an important health issue to be pursued. Based on thewide range of abnormal functional changes established after a long-termperiod in the brain produced by chronic opiate abuse, it is easy tounderstand the difficulties to re-establish the homeostatic function ofthe brain, prior to drug-intake, by the current available detoxificationtherapies. Thus, despite these therapeutic limitations, an idealdetoxification treatment must be address to meet specific medicalcriteria described as follows. Firstly, it should be directed to blockor blunt the physiological and psychological opiate dependence in orderto re-establish the homeostatic balance of those neural systemschronically dysregulated by opiate substances. Secondly, thedetoxification treatments should inhibit those pertinent physical andbehavioral changes that appear to be exacerbated during drug withdrawalinduced by therapeutic interventions, thereby resulting in a tolerableexperience and safety treatments. Additionally, it should provide acomplete suspension of the individual's drug-intake behavior, thusreorientating the addicted individuals to other alternate availablenon-pharmacological treatments (e.g., psychotherapy and counseling).Thereafter, once the complete opiate detoxification therapy is reached,the final medical goal to be approached is the prevention of subsequentrelapses to opiate abuse. Thus, from a general medical viewpoint, thetherapeutic challenges to blunt morphine/heroin addiction are enormousand, in most of cases, difficult to improve. The main obstacles faced byboth pharmacological and non pharmacological-based treatments, are thelack of an adequate number of specialized clinics or hospitals, the higheconomical costs of therapy usually billed to the patient and, mostimportantly, the absence of either patient follow-up programs (i.e.,years) or continuous clinical evaluation as well as the lack ofapplication of long-term psychotherapy support to prevent drug-relapse.In addition, another major problem facing most of the currentanti-addictive treatments against opiate abuse is the side-effecttoxicity resulting from long-term dosification of single or combinedpharmacological agents (K. Severino et al., Ann. N.Y. Acad. Sci 909:51-87, 2000). For example, methadone and buprenorphine, two long-lastingpartial agonist of the mu opioid receptor, represent the most commonsubstitution therapeutic drugs used today to blunt opiate withdrawalsyndrome or to prolong opiate abstinence (M. J. Kreek, Ann. N.Y. Acad.Sci, 909: 186-216, 2000). In addition, α₂ adrenergic receptor agonistssuch as clonidine, guanfacine and/or lofexidine represent another set ofcompounds used quite frequently in detoxification therapies toamielorate withdrawal signs and symptoms caused by the abruptsuppression of opiate drugs (M. J. Kreek, Ann. N.Y. Acad. Sci,909:186-216, 2000). However, besides of their widely use in long-termdetoxification therapies and/or treatment maintenance of abstinence,these drugs have been shown to induce several toxic side-effects. Forexample, methadone, buprenorphine and pentazocine have been reported toproduce sleep disorders, anxiety and severe cognitive and emotionalimpairment. Additionally, α₂ adrenergic receptor agonists have beenreported to produce sedation, hypotension, extreme anxiety and astheniaupon long-term administration. In addition, patients receivingopiate-substitution with methadone, may not surprisingly, show thedevelopment of signs and symptoms of opiate-dependence due that this muopioid receptor partial agonist produces same neurochemical, cellularand molecular neuroadaptative changes in the brain, as those reportedfor both morphine and heroin during opiate addiction (M. J. Kreek, Ann.N.Y. Acad. Sci, 909:186-216, 2000). Other available drugs currently usedto prolong abstinence and to relapse prevention against morphine/heroinaddiction in detoxified patients comprise the mu opioid receptorantagonists, naloxone and naltrexone. The toxic side effects often seenduring the long-term administration of these compounds are mostly due tothe blockade of the endogenous opioid transmission systems in the brain,leading to impairment of both cognitive and emotional brain functions,among many other physiological activities (M. J. Kreek, Ann. N.Y. acad.Sci, 909:186-216, 2000).

Thus far, one major conclusion drawn from the above describedpharmacological therapies currently used to approach detoxificationagainst opiate abuse including long-term maintenance treatments fordrug-withdrawal and relapse-prevention against morphine/heroinaddiction, is that none of these pharmacological treatments have shownan optimum efficacy. This conclusion is based on the fact that thesedrugs produce important toxic side-effects in patients receivinglong-term maintenance of abstinence and/or relapse-prevention (T. Kostenand D. Biegel, Expert Rev. Vaccines, 1(3): 89-97, 2002). Thus, there isan urgent need to develop and validate novel anti-addictive therapeuticstrategies, based on the synthesis, application and validation of highlyeffective new drug formulations, displaying minimum toxicity and nodetected side-effects, when pretend to be use in the long-term therapiesfor acute detoxification and long-term maintenance of morphine/heroinabstinence.

For this reason, here are given and shown all the reports and documentsconcerning the state of the art of the development and application oftechniques related to the present invention, which are detailed hereinand are also included to be used only as reference material.

In this context, different groups have designed, applied and validatedalternative therapeutic strategies in experimental animal models, whichshare a common pharmacokinetic mechanism. Thus, conversely to theclassical anti-addictive pharmacology, this latter mechanism is based onaltering the drug's pharmacokinetics by decreasing significantly orblunting the blood-brain barrier permeation of the “free” unbound drugin plasma, which ultimately represents the fraction of drug in plasmathat permeates the brain causing the high reinforcing and rewardingeffects in the addicted individual. All of these experimental approacheshave been focused to decrease significantly or prevent the blood-brainbarrier permeation of drugs of abuse, by enhancing the binding of the“free” unbound fraction of drug in plasma by specific antibodies, whichrecognize and bind with high specificity and avidity to these drugs inthe blood. As immunoglobulins (antibodies) do not normally permeate theblood-brain barrier, the plasma fraction of “free-unbound drug”, whichis the available pool of drug that permeate the blood brain barrier,interact with immunoglobulins establishing drug-antibody complexes,which ultimately decreases significantly this fraction of free-unbounddrug in plasma. This change in the drug's pharmacokinetics in plasma,leads to altered changes in drug's pharmacodynamics in the brain, thusblunting or abolishing the activity of addictive drugs on their specifictargeted neurons. These latter pharmacodynamic changes ultimately leadto blunt both the development of the reinforcing activities and therewarding pleasant effects induced by drugs of abuse in the brain. Themain pharmacokinetic property shared by most drugs of abuse, is the highblood-brain permeation activity, which represents the basic and crucialmechanism, by which most of the potent drugs of abuse produced theirhighly intense reinforcing actions in the brain, leading to thecontinuous drug-intake and drug-seeking behaviors display by individualsupon exposure to these chemical compounds. In this context, thegeneration of specific serum antibodies against drugs of abuserepresents an alternate therapeutical approach to blunt or prevent theblood-brain barrier permeation of drugs of abuse from reaching itsneuronal targets. This antibody antagonism approach preventing drug'spermeation into the brain has been shown to enhance an immunoprotectiveeffect against drug-intake and drug-seeking behaviors, as demonstratedfor cocaine, nicotine, PCP and amphetamines in rodents (see an accountof reviewed works and references therein in T. Kosten and D. Biegel,Expert Rev. Vaccines, 1(3): 89-97, 2002 and K. Kantak, Drugs, 63(4):342-252, 2003). With regard to cocaine, several research groups wereable to develop and apply different experimental strategies based on thedesign, synthesis, application and validation of several immunogenicpreparations of carrier proteins with covalently haptenized cocaine(Kantak et al., Psychopharmacology 148:251-262, 2000; Fox, B. S. et al.,Nat. Medicine, 2:1129-1132, 1996; Sparenborg et al., Therapeutics293(3): 952-961, 2000; Carrera et al. Nature, 378:727-730, 1995,Carrera, R. et al., Proc. Nat. Acad. Sci, USA, 97(11)6202-6206, 2000).Some high molecular weight proteins such as BSA and KLH have been usedas carriers to covalently link cocaine using standard chemical covalentcoupling procedures. In this way, following active immunizationprotocols in rodents, some of these immunogens have shown capabilitiesto generate low to moderate antibody titer responses against this drugof abuse in actively vaccinated animals. Moreover, other experimentalapproaches conferring immunoprotective effects against cocaine addictionhave been explored by enhancing the generation of conventional and/orcatalytic monoclonal antibodies administered during passive immunizationprotocols against this psychoactive drug in rodents (Metz et al., Proc.Natl. Acad. Sci. USA 95:10176-10180, 1998; Fox et al., Nat. Med.2:1129-1132, 1996 and Landry et al., Science, 239:1899-1901, 1993). Theimmunoprotective effects against cocaine addiction using theseimmunological-based experimental strategies have been explored bydetecting the abolishment of the drug-reinforcing behaviors in rodentsin combined pharmacological and operant-behavioral paradigms. Theseexperimental strategies share a common anti-addictive mechanism, whichrelies in the significant reduction and/or complete inhibition of theblood-brain permeation of the “free” unbound fraction of cocaine inplasma. Thus, in actively vaccinated hyperimmune animals, the fractionof “free” unbound of drug in plasma is significantly reduced, oncespecific serum antibodies in the blood bind to the psychoactive drug(Kantak et al., Psychopharmacology, 148:251-262, 2000; Carrera et al.,Proc. Natl. Acad. Sci. USA, 97(11):6202-6206; Carrera et al., Nature,378:727-730, 1995). Alternatively, monoclonal antibodies raised againstcocaine, may bind the “free” unbound fraction of cocaine after beingpassively transferred into the blood (Metz et al., Proc. Natl. Acad.Sci. USA 95:10176-10180, 1998; Fox et al., Nat. Med. 2:1129-1132, 1996,Benowitz, Pharmacol. Toxicol. 72:3-12, 1993). In both cases, the commonimmunological neutralizing mechanism, which promotes altered changes incocaine pharmacokinetics, leads to the significant decrement or completeinhibition of drug's entry into the brain, thereby decreasing orblunting the targeting of cocaine to the specific neuronal membranedopamine reuptake transporter. This latter antibody-mediated mechanisminducing altered changes in cocaine's pharmacodynamic in the brain,would lead to changes in the synaptic level of amine neurotransmitters,abolishing the evoked-dependent increase in the centralcatecholaminergic tone, normally seen after cocaine's entry into thebrain in addictive individuals. The final behavioral scenario thatresults from these altered changes in cocaine pharmacokinetics andneurochemical events is the lack of the intensified rewarding effectsinduced by this potent reinforcing drug in the brain of mammals. Thus,once cocaine has been neutralized to produce its reinforcing andrewarding effects in hyperimmune animals, the reinforcing properties ofthis drug will be lost upon a subsequent drug exposure, as demonstratedby the suppression of drug-seeking and drug-intake behaviors in suchhyperimmune vaccinated animals (rodents) seen with use of someimmunogenic conjugates of cocaine.

In summary, most of the aforementioned pre-clinical studies have shownthe feasibility of using antibody-based antagonism approach for bluntingdrug-taking and drug-seeking behaviors in rodents. However, the type ofthe carriers proteins (e.g., BSA and KLH) used in the preparation of theimmunogenic conjugates (vaccines) used in these studies preclude itspotential use in vaccine formulations for use in human immunizationprotocols (Carrera et al., Proc. Natl. Acad. Sci. USA, 2001; Carrera etal., Proc. Natl. Acad. Sci. USA, 2000; Carrera et al., Nature,378:727-730, 1995; Kantak et al., Psychopharmacology, 148:251-262, 2000;Ettinger et al., Pharmacol. Biochem. Behay. 58:215-220, 1997 and Fox,Drug and Alcohol Depend. 48:153-158, 1997). Furthermore, the synthesisof conventional andor catalytic mouse anti-cocaine monoclonal antibodiesused as potential passive immunotherapy for addition in experimentalanimals (rodents), has the main limitation in conferringimmunoprotection in a short-term period when passively administered.This is mostly due to the fast metabolic clearance of these murineimmunoglobulins from serum of passively immunized animals other thanmice (Goldsby et al., Vaccines, In: Kuby Immunology, 4^(th) ed. Freemanand Co. New Cork, N.Y., pp. 449-466, 2000). Moreover, the potential useof the available murine anti-cocaine monoclonal antibodies asimmunological therapeutic agents against cocaine addiction in humans,requires the use of DNA recombinant techniques, so as to “humanize” theFc segment of murine immunoglobulins.

Finally, the potential application of this antibody-based antagonismagainst cocaine addiction in the human is a current issue underexperimentation as a therapeutical approach. This immunopharmacologicalstrategy was initially approached through the synthesis of ananti-cocaine vaccine formulation, structurally designed for human use,where cocaine was covalently conjugated to the recombinant β-subunit ofthe cholera toxin (used as carrier protein. At pre-clinical level, thisconjugate showed moderate efficacies in triggering antibody responses inactively vaccinated rats that conferred immunoprotective effects toprevent relapse to cocaine taking-behavior in this animal (Kantak etal., Psychopharmacology, 148:251-262, 2000). Additionally, activevaccination with this immunogen in human volunteers, used to test thesafety and immunogenicity of this vaccine formulation, unfortunately,showed little promissory therapeutic effects, in this single ClinicalPhase I study (T. Kosten et al., Vaccine 2559:1-9, 2001), due to thefact that this vaccine formulation showed a poor immunogenic capacity,producing low antibody titer responses [e.g., low concentration range(μg) of specific immunoglobulin/ml of serum] in most of the vaccinatedsubjects. In addition to the aforementioned experimental limitations,new anti-cocaine vaccines developed by different groups of research, arecurrently being under study, using different carrier proteins, in orderto generate an improved immunogenicity against this psychoactive drug inboth pre-clinical and Clinical Phase I studies. Once the immunogenicproperties of these vaccine formulations are validated in humans inClinical Phase I studies, it may become available for a subsequentevaluation in Clinical Phase II studies assessing the immunoprotectingcapabilities of these vaccine formulations against cocaine addiction.For instance, it could be used Clinical Phase II studies by assessingthe enhanced long-lasting humoral-based immunoprotection against cocaineaddiction, in former drug addicts, exhibiting a prolong and controlledabstinence but challenged to the pharmacological reacquisition ofaddictive cocaine-intake behavior.

In the case of tobacco addiction, at least two immunogenic preparations(vaccines) to the reinforcing psychoactive substance, namely nicotine,have been designed for human application (see an account of reviewedworks and selected references therein in T. Kosten and D. Biegel, ExpertRev. Vaccines, 1(3): 89-97, 2002; K. Kantak, Drugs, 63(4): 341-352,2003). Pre-clinical studies have demonstrated that these two vaccineswere able to generate low to moderate serum titers of specificantibodies (i.e., 0.05-0.2 mg/ml of serum) against nicotine in activelyvaccinated rodents. Moreover, active vaccination with these immunogenicpreparations of nicotine, demonstrated to confer immunoprotectionagainst the acquisition nicotine-intake behavior in intravenousdrug-self-administration paradigms in rodents. The immunoprotectivemechanism against nicotine addiction follows same pharmacokineticmediated-mechanism described for cocaine addiction, that is, through thebinding of the “free” unbound fraction of nicotine in plasma by specificserum antibodies, which prevents the blood-brain barrier permeation ofthis drug. Clinical Phase studies performed independently by NabiPharmaceuticals (Anti-nicotine vaccine NicVAX) and Xenova PharmaceuticalGroup in Belgium, reported successful results on the evaluation of thetoxic and immunogenic properties of these two vaccine formulations. Thereports on the evaluation of the immunoprotection capabilities of thesetwo vaccine formulations against nicotine addiction in former drugaddicted volunteers in Clinical Phase II studies are expected to beready in the next two coming years.

In fact, the development of experimental strategies focused in thedesign and synthesis of immunogenic preparations and the subsequentvalidation of vaccination protocols for treating specific forms of drugaddiction, were pioneered approached for opiates such as morphine andheroin, but not for cocaine and nicotine addiction. Retrospectively, atthe beginning of the 70s, different research groups showed thefeasibility of raising a humoral immune response against these twoopiate substances using vaccination protocols in experimental animalmodels, such as the rat and the rabbit (S. Spector and C. W. Parker,Science, 168:1347-1348, 1970; S. Spector, J. Pharmacol. Exp. Ther.178:253-258, 1971; E. L. Adler and C. Liu, J. Immunol, 106:1684-1685,1971; H. Van Vunakis et al., J. Pharmacol. Exp. Ther. 180:514-521, 1972;B. H. Wainer et al., Science, 176-1143-1145, 1972; B. H. Wainer et al.,Science, 178:647-648, 1972 and B. H. Wainer et al., J. Immunol.110:667-673, 1973). These experimental approaches were focused ingenerating polyclonal antibodies against morphine, displaying distinctimmunological cross-recognition against heroin and structurally relatedopiate analogues (e.g., codeine, meperidine, and hydromorphone). Theseantibodies were generated for using in specific-designed immunoassays(i.e., radioimmunoassay and ELISA immunoenzymatic assays) to detect andmeasure morphine and related opiate substances in biological fluids fromhumans. These studies showed, for the first time, the successfulachievement on the design and validation of the covalent condensation ofthe exposed free 3- and 6-hydroxyl groups in the phenantrenic ring ofthe morphine molecule to carrier proteins such as BSA, using standardorganic chemistry procedures (procedures that are still used whenapproaching chemical synthesis of such immunogenic conjugates). Inaddition, it is worth to mention that none of these chemical procedureswere never reported and claimed in patent registries and they are mostlyconsidered as classical chemical procedures in textbooks of organicchemistry, when describing the covalent linkage of the free 3- and6-hydroxyl groups of the phenantrenic ring of morphine to carrierproteins. In such context, two structural intermediate products frommorphine were successfully synthesized by different research groups andused for the development of vaccine formulations, namely, the3-ortho-morphine-carboxymethyl-ether product (3-O-carboxymethylmorphine,see example 2) and the morphine-6-hemisuccinate (see example 3) (S.Spector and C. W. Parker, Science, 168:1347, 1970; S. J. Spector, J.Pharmacol. Exp. Ther. 178:253, 1971; H. Van Vunakis et al., J.Pharmacol. Exp. Ther. 180:514, 1972; and S. Gross et al.,Immunochemistry, 11:453-456, 1974). With regard to the aforementionedintermediate derivatives of morphine used to develop vaccineformulations, two identical patent registries published on Sep. 13, 1991(CH 678394 A5) and May 15, 1996 (EP 0 496 839 B1) by Erich Hugo Cerny,claim invention on the structural synthesis of novel anti-morphinevaccine formulations. However, it's worth to mention, that both of thesepatent registries reveal no real novelty or invention regarding thesynthesis of the therapeutic vaccine formulations claimed. This argumentis based on that both patent registries describe the same standardsynthetic procedures previously reported to generate the intermediate3-O-carboxy-methyl-morphine derivative used to covalently conjugate theKLH-carrier protein. In both instances, they used morphine-based and thesodium beta-chloroacetate and absolute alcohol as reagents in thereaction mixture. The other synthetic intermediate derivative used toactivate the covalent linkage between morphine and carrier proteins, isthe morphine succinyl ester linked to the free 6-hydroxyl group of thephenantrenic ring-structure of the morphine molecule, namely,morphine-6-hemisuccinate, (see example 3) (originally reported by B. H.Wainer et al., Science, 176:1143, 1972, A. Akbarzadeh et al.,Biotechnol. Appl. Biochem, 30:139-145, 1999). In same context to theaforementioned synthetic procedures used to generate the3-O-carboxymethylmorphine derivative for synthetizing vaccineformulations, an anti-morphine vaccine patent registry released fromChina (CN1196955), was unjustified granted from our own perspective, toHan Ying et al., on Oct. 28, 1998. These authors claim innovation andnovelty regarding the synthetic procedures and the structuralformulations of vaccine preparations to different opiate drugs, besidesmorphine, using same standard methods to synthesizemorphine-6-hemisuccinate derivative, as previously reported (see in B.H. Wainer et al., Science, 176:1143, 1972, A. Akbarzadeh et al.,Biotechnol. Appl. Biochem, 30:139-145, 1999). These authors used thisintermediate derivative to haptenize morphine to BSA as carrier protein.

The structural design and synthesis of different immunogenicformulations, where morphine has been haptenized to carrier proteinssuch as KLH and BSA, represented the basis by which authors haveinvariably used chemical procedures to link covalently the intermediatederivatives of morphine 3-O-carboxymethylmorphine andmorphine-6-hemisuccinate to these carrier proteins (as previouslyoutlined in the experimental studies described above, including theaforementioned patent registries), using as cross-linker thehomobifunctional chemical reagent,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The EDC reacts withthe available free carboxyl groups exposed in either the3-O-carboxymethylmorphine or morphine-6-hemisuccinate derivatives, thusforming the corresponding two O-acylurea by-products, which arechemically reactive to generate covalent amide bonds with the epsilon(ε)-amino groups in the lateral chain of lysine residues of either BSAor KLH (see example 4).

The aforementioned studies demonstrating the feasibility to generate ahumoral immune response against morphine and its structural cognatesemisynthetic opiate, heroin, led to a pioneer study reported nearly 30years ago by Bonese (K. F. Bonese et al. Nature, 0 252:708-710, 1974).This study was in fact the pioneer report demonstrating that activevaccination with an immunogenic morphine conjugate in a singleexperimental animal, the non-human primate Macacus rhesus, was able togenerate a protective humoral-mediated immune response that blunt theaddictive self-administration behavior to heroin. The synthesis of thisimmunogenic conjugate was achieved by covalently haptenizing morphine tothe BSA through a stable ester bond formed by the condensation of thesuccinic anhydride and the 6-hydroxyl group in the phenantrenicstructure of the morphine molecule. The synthesized intermediatederivative, morphine-6-hemisuccinyl, was then covalently linked to the1-ethyl-3-(3-Dimethylaminopropyl)carbodiimide (EDC) reagent, thusobtaining the complete immunogenic conjugate. The repeated subcutaneousinjection of this immunogen into the primate triggered a humoralimmunological response with specific morphine antibodies, whichdisplayed cross-recognition for heroin. Additionally, the activevaccination approach with this conjugate demonstrated to be an effectiveprocedure to blunt the re-acquisition of the intravenousself-administration behavior to heroin in this single primate,previously trained to self-administer different dose-units of thisopiate. Although this pioneer report showed the first successfulantibody-based antagonism procedure to blunt heroin-intake behavior inthe primate, it was never patented and developed for clinical use.Similarly, no further experimental studies related to the design,synthesis and validation of further novel structuralanti-morphine/heroin vaccine formulations using carrier proteinssuitable for human vaccination were developed, basically due to the factthat BSA is not a licensed carrier protein for such experimentalpurposes. The main reason that these immunopharmacological studies werenever approached in humans with suitable immunogens for morphine orheroin could be due, at least in part, to the simultaneous andcontinuous development of other neuropharmacological agents used totreat morphine-heroin addiction. For instance, synthetic drugs thatdisplay weak and partial agonist activities on the mu opioid receptor(i.e., methadone and buprenorphine) and other drugs which displayantagonist activities on opioid receptors (i.e., naltrexone andnaloxone). All these drugs are currently used for preventing relapse toopiate addiction.

Based on the aforementioned reports of experimental vaccines againstmorphine/heroin addiction, which never approached human vaccination,preliminar experimental studies conducted by our research group servedas a basis, for the development of the present invention of the bivalentvaccine formulation against morphine-heroin addiction. These experimentsdescribe the design, synthesis and evaluation of the immunogenicityinduced by different synthesized structural models of a new generationof vaccines against anti-morphine/heroin (B. Anton and P. Leff., 31^(st)Annual meeting of the Society for Neuroscience. San Diego, Calif. Nov.10-16, 2001). Such structural formulations of vaccines were initiallysynthesized by linking covalently the morphine-6-hemisuccinate (M-6-H)intermediate derivative with several carrier proteins such as BSA, KLHand the recombinant cholera toxin-β-subunit protein. These couplingreactions used standard crosslinking procedures for linking themorphine-6-hemisuccinate (M-6-H) intermediate derivative to the1-ethyl-3-(3-Dimethylaminopropyl)-carbodiimide (EDC) reagent. Thesepreliminary experimental data gathered from such studies made possiblethe identification of candidate carrier proteins for covalenthaptenization of morphine. It is worth to mention that this work wasonly presented in a slide session at the aforementioned InternationalNeuroscience Meeting.

Furthermore, it did not show any information concerning experimentaldata related to the design of the structural molecular models ofimmunogens, methodologies describing the synthesis, purification,application and dosification procedures of these new vaccines. Moreover,no references or descriptions of the validation of the anti-addictiveeffects against morphine-heroin were also made, which are disclosed inthe present invention of the therapeutic bivalent morphine-heroinvaccine formulation against the addiction to these opiate substances.

In addition to the pioneer study reported by Bonese and co-workers, whodemonstrated the efficacy of the active vaccination with BSA-morphine toblunt the addictive self-taking behavior of heroin in a single primate,other research groups explored the immunoprotective effects of thepassive immunization against morphine, using behavioral paradigms ofintravenous self-administration of heroin in rodents (P. R. Pentel etal., Pharmacol. Biochem. and Behavior, 9:347-352, 1991). From apotential therapeutical viewpoint, a passive immunoprotection procedureagainst morphine and heroin addiction has practical limitations toprolong and maintain abstinence to opiate drugs in humans on a long-termbasis, as opposed to the active immunization procedures. Theselimitations are based on some practical observations derived fromexperimental results of passive immunoprotection paradigms (R. A.Goldsby, Vaccines. In: Kuby Immunology, 4^(th) ed. Freeman and Co, NewCork, N.Y., pp. 449-466, 2001). These data have demonstrated therelatively short biological half-life of murine monoclonal antibodies(3-23 days, depending on the immunoglobulin class and isotype) afterbeing administered in vivo into different experimental animals. Thus,immunoprotection conferred via passive administration of murinemonoclonal antibodies into non-murine hosts is usually short-lived.Moreover, as both monoclonal and polyclonal antibodies used in passiveimmunization therapies are commonly produced from different animalspecies (e.g., mouse, rabbit, etc.), such immunoglobulins are usuallyrecognized as foreign antigenic molecules when injected into humansubjects. In this context, passive immunization of patients with suchimmunoglobulins would trigger a rapid humoral immunological responseagainst these molecules, which ultimately result in a bluntedantibody-mediated neutralizing responses and reduced half-life of thesetypes of immunoglobulins in plasma. Thus, once primed such antigenicresponses against heterologous antibodies in passively immunizedsubjects, the subsequent administration of these types ofimmunoglobulins would lead to the development of abnormal immunologicalresponses of hypersensibility upon repeated passive administration ofsuch molecules.

AIMS AND ADVANTAGES OF THE INVENTION

Based on the aforementioned background regarding theanti-addictive-based therapies against opiate abuse, specificallyagainst morphine and heroin addiction, we can conclude that noefficacious and atoxic drugs are yet available in humans for treatingand maintain prolong abstinence and relapse prevention from addictiveopiate-intake behaviors. Thus far, reasons exist to justify the currentneed for the development, application and validation of combined newdrugs and therapeutic strategies to maintain prolonged abstinence withefficacy for preventing relapse to addictive drug-intake behaviors tohighly addictive opiate drugs such as heroin and morphine in the humans.

As previously mentioned, the previous experimental studies reporting thepre-clinical evaluation of different immunological strategies againstdrug addiction in animal models, support the potential therapeuticapproach of the different immunoprotective strategies against cocaine,nicotine and opiate addiction (specifically to both morphine andheroin). This strategies include new pharmacological treatments based onantibody antagonism for the maintenance of prolong abstinence and/orprevention of relapse to drug intake and drug addiction to theaforementioned substances in the humans. In fact, the most importantlegacy of these immunoprotective studies against drug addiction, is theidentification of the critical experimental achievements from bothpre-clinical and clinical Phase studies, which ultimately would lead tothe potential use and validation of these immunological strategies inhumans to maintain prolong abstinence and/or to relapse preventionduring addictive disorders to illegal drugs of abuse.

In this context, we can mentioned the following experimentalrequirements to be meet: a) design and synthesis of structuralformulations of anti-addictive vaccines where the haptenic drug isstructurally coupled via very stable covalent links (i.e., amide), usingbifunctional chemical compounds with low structural complexity andimmunogenicity that enhance the covalent cross slinking of the haptenicdrug with licensed carrier proteins used in vaccination protocols in thehumans; b) such proteins should display proven atoxicity, and should beable to confer a very high immunogenicity to the haptenic drug when thedrug-protein conjugate is administered in active immunization protocols;c) systematic evaluation of the humoral immune response to the haptenicdrug conjugated to the carrier protein, so that functional parameters oftriggered specific antibodies such its titers, specificity and aviditycan be properly characterized after application of the immunoconjugatein ad hoc active vaccination protocols; d) systematic monitoring of thehumoral immune response during the active vaccination protocols with theimmunogenic conjugate containing the haptenized addictive drug, so as toidentify and assess the establishment of a long-term and stable humoralmemory response against the antigenic drug after completion of theactive immunization protocols; e) assessing of the capacity and efficacyof these new therapeutic vaccine formulations against drug addictions(i.e., morphine-heroin) with proven capacities to confer long-termimmunoprotection against addictive drugs. These vaccines should exhibita good therapeutic index to prevent the reacquisition of the addictivebehaviors in detoxified and abstinent subjects.

The present invention relates to the design, synthesis, purification,application and validation of a novel structural model of vaccineagainst both morphine and heroin addiction, that fulfills all theaforementioned structural and functional requirements. The detaileddescription of the synthesis procedures and the structure of thiscarrier protein-morphine conjugate, are disclosed in the presentinvention. Other information also disclosed therein are its use inparadigms of active immunization in the rodent, the monitoring andcharacterization of the development of the humoral immune response afterboosting, as well as the titers and the antibody specificity generatedagainst the haptenized drug. Additionally, experimental data are alsodisclosed showing the proven efficacy of the present invention of thetherapeutic formulation of a novel bivalent vaccine againstmorphine/heroin to induce a robust humoral immune response of high andsustained serum antibody titers against morphine, with equivalentspecificity for heroin, in detoxified and abstinent subjects addicted tothese opiate substances. Such antibodies can efficiently antagonize(block) the reacquisition of the addictive drug-intake and drug-seekingbehaviors, in addition to prolong the abstinence state against these twoopiate substances in hyperimmune subjects challenged to drug'sreacquisition in a standard addictive intravenous self-administrationparadigm of these two opiate substances in the rat.

The first aim of the present invention is to disclose the method ofsynthesis and the structural formulation of a novel morphine immunogen,which has the following functional and structural advantages neverpresented in any other previously synthesized morphine/heroin vaccineformulations: a) covalent morphine haptenization to the tetanus toxoid,a carrier protein licensed in active vaccination protocols in the humanwith proven capacity to confer a very high immunogenicity to haptenmolecules of low molecular mass; b) covalent morphine haptenization tothe tetanus toxoid, through the sequential use and covalent linkage oftwo different crosslinking reagents, which enhances the synthesis of along and low immunogenic spacer linker-arm placed between the carrierprotein and the haptenized drug; c) this novel morphine immunogen wasdosified in active vaccination protocols in subjects, and showed aproven efficacy to generate a robust and sustained humoral immuneresponse characterized by highly specific serum antibodies withequivalent specificities against morphine and its structurally relatedand highly addictive opiate analogue, heroin.

Another aim of the present invention is to disclose and validate anactive immunization paradigm using the aforementioned carrierprotein-morphine conjugate, for optimizing a robust and sustainedhumoral immune response with very high anti-hapten antibody titers withan established long-term immune memory response.

Another aim of the present invention deals with the demonstration foroptimizating the antibody titer and specificity, in order to demonstrateits functional capacity for cross-recognizing heroin, but not otheropiate medications structurally-related to morphine and/or severalendogenous opioid peptides produced in the brain.

Another aim of the present invention concerns with the validation on theuse of this immunogen, as a novel pharmaceutical composition ortherapeutical formulation to confer immunoprotection against there-acquisition of addictive morphineheroin-intake behaviors and for themaintenance of prolonged abstinence in experimental subjects previouslydetoxified from either morphine or heroin addiction.

An additional aim of the present invention discloses the synthesis andthe molecular structure of a therapeutic anti-morphine/heroin vaccineformulation, validated in pre-clinic studies in the rodent, where thisvaccine formulation containing the tetanus toxoid used as theimmunogenic carrier protein to covalently haptenized morphine, providesits potential use to evaluate its therapeutical effects in clinicalphases studies, by conferring a long-term immunoprotection, maintenanceof prolonged abstinence and relapse prevention in detoxified subjectsfrom either morphine or heroin addiction.

A final aim of the present invention discloses the general methodologyused to design, develop, apply and validate an efficient and atoxictherapy, whose mechanism of action differed from the available currentclassical therapeutic compounds, by enhancing pharmacokinetic changes ofthe aforementioned opiate drugs, thereby reducing efficiently theirblood-brain barrier permeation, once they have been administered to ahyperimmune subjects, previously vaccinated against these two opiatedrugs of abuse.

Collectively, the present invention discloses and provides a fulldescription of the methodology and processes required to prepareintermediate derivatives for the synthesis of a morphine/heroin vaccine,pharmaceutical compositions or therapeutic formulations, includingmethods and therapeutical uses against morphine-heroin addiction.

BRIEF DESCRIPTION OF THE FIGURES

Other features and advantages of the present invention will be evidentfrom the specific aims and preferred modalities described in the claimsdisclosed and footnotes accompanying drawings or figures, wherein:

FIG. 1, depicts a representative immunoenzymatic antibody capture ELISAassay showing the robust humoral immune response induced by the noveltetanus toxoid-morphine immunogen characterized by high serum antibodytiters generated against this opiate drug;

FIG. 2, shows the a representative plot which depicts the monitoring ofthe humoral immune response of serum titers of morphine/heroinantibodies in the rat, quantified through antibody capture ELISA assays,along four consecutive reboosts with the novel tetanus toxoid-morphineduring the active vaccination schedule in the rat;

FIG. 3, shows a representative plot of antibody capture immunoenzymaticELISA assays used to monitor the behavior of the humoral immune responseafter the last re-immunization (fourth) with the new tetanustoxoid-morphine immunogen;

FIG. 4, depicts a representative plot of antibody captureimmunoenzymatic ELISA assays used to monitor the recovery of themorphine/heroin specific antibody titers induced after a subsequentfifth boost with the new tetanus toxoid-morphine immunogen;

FIG. 5, depicts a competitive immunoenzymatic ELISA assay used toevaluate potential cross-recognition of the anti-morphine/heroin serumantibodies for different structurally-related analogues to morphine andheroin, used in classical anti-addictive therapy against these twoopiate compounds, including the biotransformation metabolites from thesetwo drugs, as well as different endogenous opioid neuropeptides involvedin the regulation of different physiological and neural bioactivities inthe CNS of mammals;

FIG. 6, shows the immunoprotective effect induced by the activevaccination with the tetanus toxoid-morphine immunogen in the rat, forblunting the intravenous self-administration behavior to heroin in thesame animal and finally;

FIG. 7, shows the immunoprotective effect induced by active vaccinationwith the tetanus toxoid-morphine immunogen in the rat for blunting theintravenous self-administration behavior to morphine in the same animal.

FIG. 8 depicts the preparation of the intermediate derivative ofEDC-morphine-6-hemisuccinate. FIG. 8A depicts synthesis of morphine basefrom a commercial sulfate salt. FIG. 8B depicts recrystallization of theM-6-H residue. FIG. 8C depicts the covalent conjugation of M-6-H withthe carrier protein.

FIGS. 9A-9C depict the reaction process used for preparation of thetetanus toxoid-intermediate derivative used as carrier protein (CP)covalently condensed with the N-α-trifluoroacetylcaproyloxy)-succinimideester (TFCS): CP-TFCS complex.

FIG. 10A depicts covalent condensation of the intermediate product ofmorphine, the EDC-(M-6-H) to the tetanus toxoid-TFCS complex. FIG. 10Bdepicts the reaction process between the free carboxyl groups exposed atthe end of the EDC-(M-6-H) conjugate and the unprotected free aminogroups from the TFCS reagent linked to the tetanus toxoid.

FIG. 11 depicts the molecular structure of the bivalent vaccine againstmorphine-heroin addiction.

FIGS. 12A and 12B depict an alternate model of synthesizing anEDC-3-O-carboxymethylmorphine derivative.

FIG. 13 depicts the molecular formular of an alternate bivalentanti-morphine-heroin vaccine.

DETAILED DESCRIPTION OF THE INVENTION

The scientific literature referred to in this section describes in fulldetails the available information to skilled persons in this field. Thepresent invention discloses and provides a therapeutical treatment formorphine and heroin addiction. This therapy is based on thepharmacological principle which describes the active vaccination with anovel structural formulation of a carrier protein-haptenic drugconjugate against these two opiates in subjects previously addicted andsubsequently detoxified. The chemical composition of the therapeuticconjugate of the present invention consists of morphine as haptenic drugand the tetanus toxoid as the highly immunogenic carrier protein, beingthis latter carrier protein a highly immunogenic licensed protein usedin human vaccination protocols. This highly immunogenicmorphine-conjugate is able to stimulate the generation of high andsustained serum antibody titers against haptenized morphine whendetoxified individuals against opiate addiction receive this therapeuticformulation. Thus, the use and application of adequate activeimmunization protocols, triggers the synthesis and enhances thegeneration of high serum anti-morphine antibody titers that recognizeand bind with high specificity and avidity to the “free” unboundfraction of drug in plasma, after a subsequent re-exposure of the drug.This process eventually leads to a significant neutralization and/orprevention of the blood-brain barrier permeation of the opiate drug,thereby decreasing or preventing significantly the reinforcingproperties of opiates in the brain. Thus, morphine and/or heroin areneutralized before reaching the brain tissue, and thereby, thedetoxified addicted subject is not rewarded by the reinforcingpharmacological properties of these two drugs, which ultimatelyrepresent the underlying “pharmacological driving system” by which thesetwo opiates enhance their reinforcing drug activities in the brainrewarding pathways. The active immunization paradigm inducing theneutralizing activity of these opiates occurs over a long-term period(i.e., 3-6 months) in vaccinated subjects treated with the bivalentvaccine against morphine-heroin addiction of the present invention. Thisis mostly due to the long-time course activity of the humoral immuneresponse neutralizing these opiate drugs in plasma, mediated throughoutthe specific serum antibodies raised against the haptenized drug. Inthis context, it is expected that the established long-term stability ofthe immune response, mediated through the generation of highanti-haptenic-drug antibody titers, induced by the therapeuticcomposition of the present invention, represents an efficientimmunogenic mechanism to maintain prolonged abstinence and/or preventrelapse to morphine and heroin addiction in previously detoxifiedsubjects. Furthermore, the therapeutical vaccination approach againstmorphine/heroin addiction of the present invention is compatible withother therapies currently used to maintain prolonged abstinence and/orrelapse prevention to opiate addiction. In this context, a large numberof pharmacological agents used for these therapeutical purposes, such asmethadone, buprenorphine, naloxone, naltrexone, etc., comprise amongmany other listed pharmacological drugs, the most selected therapeuticalcompounds used in clinics, which can be used simultaneously with thevaccination therapy discloses in the present invention.

The aim of the aforementioned parameters and the following exampleslisted below are shown to illustrate the particular issues required tocarry out and perform the present invention and it should not beconsidered as limiting factors of the protective pursuit of the same.

EXAMPLES 1. Schematic Representation of the Molecular Structure of theChemical Commercial Formulation of Morphine Used as Hapten for thePreparation of the Bivalent Vaccine Against Morphine-Heroin Addiction

The chemical commercial formulation (Sigma-Aldrich) of the pentahydratedmorphine-sulfate salt (MW 758.8, C₃₄H₄₀N₂O₁₀S) was used as the startingcompound for synthesis of morphine base (see below, paragraph (a), underthe section describing “A REACTION PROCESS FOR THE PREPARATION OFINTERMEDIATE DERIVATIVES USED FOR THE SYNTHESIS OF THE BIVALENT VACCINEAGAINST MORPHINE-HEROIN ADDICTION”) and then for the synthesis of theintermediate derivative morphine-6-hemisuccinate. This latterintermediate derivative was subsequently haptenized to the free epsilonamino groups from the lateral chain of exposed lysine residues in thetetanus toxoid through the sequential covalent cross-linking with thehomo- and hetero-bifunctional cross-linking reagents, EDC and TFCS,respectively.

2. Schematic Representation of the Structural Formulation of the3-O-Carboxy-Methyl-Morphine Intermediate Derivative

This intermediate derivative of morphine has been synthesized and usedby several groups of researchers and it was also used in the presentinvention for the covalent haptenization of morphine to the tetanustoxoid, as an alternative bivalent vaccine against morphine-heroinaddiction;

3. Schematic Representation of the Structural Formulation of theIntermediate Derivative Morphine-6-Hemisuccinate

This intermediate derivative of morphine has been synthesized and usedby several groups of researchers and used in the present invention ofthe bivalent vaccine against morphine-heroin addiction for the covalenthaptenization of this opiate substance to the tetanus toxoid;

4. Schematic Representation of the Structural Formulation of the3-O-Carboxy-methyl-morphine and Morphine-6-Hemisuccinate IntermediateDerivatives Covalently Condensed to the1-Ethyl-3-(3-Dimethylaminopropyl)Carbodiimide (EDC)

The chemical reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)has been used by several researchers in covalent haptenization reactionsof the 3-O-carboxymethylmorphine intermediate derivative to carrierproteins such as KLH and BSA. The EDC was also used for the covalenthaptenization of the intermediate derivative morphine-6-hemisuccinate tothe intermediate product complex tetanus toxoid-TFCS in the presentinvention of the bivalent vaccine against morphine-heroin addiction (seethe reaction schemes of chemical synthesis in examples 5 (a-c) and 7 (aand b).

Synthesis of the Bivalent Vaccine Against Morphine-Heroin Addiction

The synthesis of the immunogenic conjugate of morphine in the presentinvention requires the initial chemical modification of the morphinemolecule to generate a high reactive structural derivative of thisopiate which provides free reactive carboxyl groups, used to covalentcross-link two heterobifunctional reagents used to form the chemicalstructure of the spacer linker arm which bind to the epsilon aminogroups of the lateral chain of exposed lysine residues in the tetanustoxoid, the carrier protein used in the present invention (see example6). The coupling chemistry procedures used to modify the structure andactivate both 3 and 6 reactive hydroxyl groups of the phenantrenic ringof the morphine molecule in order to crosslink heterobifunctionalreagents are very scarced (Robert T. Morrison and Robert N. Boyd,Organic Chemistry, 7^(th) Ed., 2003), and only very few methods usingthis chemical synthesizing approaches have been reported. In thiscontext, from the beginning of the 70s, different research groups (B.Wainer et al., Science 176: 1143-1145, 1972; B. Wainer et al., Science178: 647. 1972; B. Wainer et al., J. Immunol. 110(3):667-673, 1973;Wainer et al., Nature, 241:537-538, 1973 and B. Hill et al., J. Immunol.114:1363-1368, 1975); reported a non-patented, classical chemical-basedmethodology, found today in organic chemistry textbooks, which usessuccinic anhydride as reagent to modify the reactive 6-hydroxyl group ofthe phenantrenic ring structure of the morphine molecule. This primarymorphine intermediate, referred to as morphine-6-hemisuccinate (seeexample 3, and FIG. 8B differs from morphine in its highly reactive freecarboxylic acid of the succinyl-ester group (previously linked to themorphine molecule) which can be covalently linked (see FIG. 8A tohomobifunctional cross-linking reagents such as the1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (see example 3, andFIG. 8C. This reagent has been widely used in covalent crosslinkingchemical reactions for the covalent condensation of free functionalamino and carboxyl groups from donor molecules (S. Hockfield et al.,Molecular Probes of the Nervous System: Selected Methods for Antibodyand Nucleic Acid Probes, Vol. 1, Cold Spring Harbor Laboratory Press,New Cork, 1993).

As morphine is not an immunogenic molecule by itself, the generation ofhumoral immune responses with high titers of specific antibodies againstmolecules of relative low structural complexity as this opiaterepresents a serious methodological challenge. In the present invention,the structure of the morphine conjugate was initially designed andfollowed by the synthesis of morphine-6-hemisuccinate intermediatederivative (FIG. 8B) which in turn was covalently haptenized to lysineresidues in the tetanus toxoid, used as carrier protein, via thesequential synthesis of a spacer-linker arm, structurally conformed bythe chemical condensation of the1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (referred to as thecommercial compound EDC, see example 5) and theN-(ε-trifluoracetylcaproyloxy)-succinimide-ester (referred to as thecommercial compound TFCS, see example 6). This procedure, based on thecovalent condensation of haptenized morphine to tetanus toxoid via thislong spacer linker arm, formed by the covalent condensation of EDC andTFCS reagents, allows the structural preservation of the morphinemolecule once haptenized with the tetanus toxoid. In this structuralcontext, it is expected that the morphine remains fixed to the carrierprotein in such a way that it could keeps its original stericconfiguration. This, in principle, could facilitate that other freedomains and/or reactive groups within the morphine molecule, other thanthe active domains of the opiate drug contributing to the covalenthaptenization with the carrier protein, it could be exposed andcontribute as the predominant antigenic domains or antigenicdeterminants in the native drug molecule recognized by the humoralimmune response. Additionally, the chemical nature of thehydrocarbonated structure of the spacer-linker arm (see example 6)confers to this carbon backbone a complete inert structure to anychemical reactivity, and thereby, a very low immunogenicity per se.These structural and functional properties conferred by thehydrocarbonated linker arm contributes could contribute to theimmunopredominant epitogenic role of morphine in the structuralformulation of the present invention of the bivalent vaccine againstmorphine-heroin addiction. Thus, the above proposed capabilities,structural and functional advantages of the present invention aresupported by the experimental results showing its high efficacy toproduce a strong humoral immune response (see FIGS. 1 and 2), with highand sustained specific serum antibody titers (see FIGS. 3 and 4) thatcross-recognize with equivalent specificity non-haptenized morphine,including the structural opiate analogues, heroin and the endogenousopiate metabolites, such as the 6-monoacetylmorphine and their active(addictive) glucuronide by-products (i.e. morphine-3-6-glucuronides)(see FIG. 5). The most plausible explanation for the specificity of thishumoral immune response triggered by our novel vaccine model againstthese opiate compounds is based on the antigenic presentation ofdifferent structural domains of morphine to the immune system. Thisappears to be due to the structural length of the spacer linker arm thatseparate morphine from the tetanus toxoid in such a way that allows theimmune system to react against to specific antigenic determinants of thephenantrenic structure of morphine shared by other structural opiateanalogues and its endogenous metabolites as well (i.e., heroin andmorphine-3-6-glucuronides).

5. Procedures and Reactions Used for the Preparation of the IntermediateDerivative of Morphine Required for the Synthesis of the BivalentVaccine Against Morphine-Heroin Addiction

a) Initial Preparation of Morphine Base from the Pentahydrated MorphineSulfate Salt (a Commercially Available Chemical Formulation ofMorphine).

Morphine base (FIG. 8A) was synthesized from the commercial sulfate saltof this opiate substance (Sigma-Aldrich), according to a classicalchemical procedure reported in 1972 by E. J. Simon (E. J. Simon et al.,Proc. Natl. Acad. Sci. USA, 69: 1835-1837, 1972). This reaction wascarried out as follows; 64 mg of morphine sulfate/ml were dissolved indistilled water at room temperature under constant stirring. The pH ofthis solution was adjusted at 8.0 with NH₄OH. The morphine base wassubsequently crystallized through precipitation at pH 8, filtered anddried through evaporation at 60° C. under vacuum conditions.

b) Preparation of the Intermediate Derivative Morphine-6-Hemisuccinate(M-6-H).

The M-6-H compound was prepared from morphine base, according to thefollowing protocol and subject to modifications from standard pioneerprocedures reported in the 70s by B. Wainer et al., Science,176-1143-1145, 1972; B. H. Wainer et al., Science, 178:647, 1972, B.Wainer et al., J. Immunol. 110 (3):667-673, 1973; Wainer et al., Nature,241:537-538, 1973 and B. Hill et al., J. Immunol. 114:1363-1368, 1975.The chemical reaction procedure was performed as follows; for each gramof morphine-based, 2 grams of succinic anhydride (Sigma-Aldrich) wasadded to the reacting mixture, followed by incubation with 20 ml ofpyridine or dry benzene under continuous reflux in a glass flask. Afterwarming the reacting mixture for up to 6 hours at reflux temperature(70-80° C.), the reaction mixture was slowly cooled at room temperatureand the excess of pyridine or benzene was decanted. The rest of theselatter organic components were evaporated using a continuous nitrogenstream under reduced pressure, producing a dry product residuerepresented by M-6-H. This product was exposed to 10 times-washed outperiods with 60% ethanol in distilled water to achieve recrystallizationof the M-6-H residue (FIG. 8B). The percentage yield of the product wasquantified by a standard analytical method, using thin layerchromatography analysis (TLC, the initials of the abbreviatedconventional nomenclature of this procedure) (B. Wainer et al., Science176:1143-1145, 1972). This method was approached as follows; 100 μg ofthe synthetic M-6-H residue and an equivalent amount of morphine base(compound used as reference control) were dissolved in the solventsystem of ethyl acetate:methanol:ammonium hydroxide (85:10:5, v:v:v),followed by sampling 1 μl/lane, dried at room temperature and runned inthe silica thin layer chromatography matrix with the aforementionedsolvent system. After the compounds have been chromatographicallyrunned, the silica thin layer is exposed to UV lamp stimulation at 285nm, (this wavelength is normally used to excite the chromophorerepresented by the phenolic ring of the phenantrenic structure in thefree morphine and M-6-H, respectively). In this context, the TLC profileof the synthetic M-6-H residue exhibited a relative mobility coefficient(Rf, its conventional abbreviated initials in English [Retentionfactor]) of about 0.1-0.15, whereas the morphine-free-based exhibited alarger Rf of about 0.3-0.4. The average yield of the M-6-H product in astandard synthetic reaction was always approximately 95% or more.

c) Preparation of the Intermediate Derivative ofEDC-Morphine-6-Hemisuccinate.

To achieve the covalent haptenization of M-6-H with the carrier protein,the intermediate derivative M-6-H was covalently conjugated through itsthe succinyl-free carboxyl group to the homobifunctional covalentcrosslinker reagent, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(Pierce) (see FIG. 8C), according to the standard protocol described bythe manufacturer (Pierce). During this condensing reaction, the excessof EDC that did not react with the succinyl-carboxylic group is rapidlyhydrolyzed to a non-reactive intermediate derivative compound, due tothe high unstability of this reagent when placed in an aqueous solution.Thus, a standard coupling reaction consisted in mixing 100 mg of EDC toeach 100 mg of M-6-H dissolved in 100 ml of distilled H₂O, at pH 5.5,adjusted with a 1N HCl solution. The reaction mixture is then incubatedat 37° C. for 2 hours under constant stirring. Under these conditions, ayield of approximately 98% of EDC-(M-6-H) product is regularly obtainedunder these coupling conditions. This estimation was obtained by thetitrating the free carboxylic groups of equivalent samples of both theM-6-H product, used as control, and the EDC-(M-6-H) product with 1N NaOHsolution, a standard biochemical procedure normally used to verify thepresence of free carboxylic groups at pK values of around 4.2. Thisreaction procedure generates optimum yields of synthetic EDC-(M-6-H)product, which is usually unstable in aqueous solutions and thereby,requires to be reacted rapidly with amino groups from the tetanustoxoid-TFCS intermediate derivative, whose synthesis is disclosed in thepresent invention of the bivalent vaccine against morphine-heroinaddiction, and serves as the carrier protein of same invention.

6. Reaction Process Used for Preparation of the TetanusToxoid-Intermediate Derivative Used as Carrier Protein (CP) CovalentlyCondensed with the N-(ε-Trifluoroacetylcaproyloxy)-Succinimide Ester(TFCS): CP-TFCS Complex

The tetanus toxoid preparation used as carrier protein (CP) in thepresent invention of the bivalent vaccine against morphine-heroinaddiction, had certified degree of purity (≧98%) and a total lack oftoxicity. This protein preparation is formed by the H-polypeptidesubunit, which contains approximately 858 amino acid residues, withmolecular mass of about 100 kDa. This protein subunit obtained throughstandard DNA recombination techniques, is encoded by the Clostridiumtetani gene, which produces the native bacterial tetanus toxin, andcontains only 68 copies of lysine residues along its primary aminoacidsequence of the H-polypeptide toxoid protein, whereas the native tetanustoxin consists of 1313 amino acid protein, rendering its high molecularmass of 150,700 daltons (150.7 kD. In order to achieve the synthesis ofthe tetanus toxoid-TFCS reactive intermediate, the ε-amino sites ofexposed lysine residues in this protein are covalently conjugated to theN-(ε-trifluoroacetylcaproyloxy)-succinimide ester (TFCS, Pierce) (seeFIGS. 9A-9C). The TFCS is a heterobifunctional covalent cross-linkerreagent used to conjugate, at pH 7-7.5, free ε-amino groups in thelateral chain of exposed lysine residues from high molecular massproteins, via its N-hydroxysuccinimide-ester active site. Thus, thisreaction enhances the synthesis of the tetanus toxoid-TFCS intermediatederivative through the formation of stable amide bonds. The reactionprocedure used to synthesize the tetanus toxoid-TFCS conjugate as anintermediate step required for the synthesis of the present invention ofthe bivalent vaccine against morphine-heroin addiction is described inFIG. 9B. The coupling procedures and experimental conditions used tocarry out a typical synthesis of this protein carrier-TFCS complexconsisted in the initial preparation of 40 mg/ml of a TFCS-stocksolution (134 mM) (prepared in fresh and always immediately before use)dissolved in a solution containing 10-20% DMSO/90-80% deionized H₂O(v:v). The TFCS reagent is immediately mixed with the tetanus toxoidprotein, in a molar excess ratio of TFCS 10-20 fold with respect to thetoxoid itself. Thus, for example, a typical reaction consists in themixture of 100 mg (0.5 mM) of tetanus toxoid dissolved in 4 ml of asolution containing phosphate-buffered saline solution (PBS=0.1 M ofPB/0.15 mM NaCl, pH 7.2) with 50 of the TFCS stock solution (the finalconcentration of TFCS and DMSO achieved in the mixture of tetanustoxoid-TFCS solution is 6.7 mM and 0.5-1%, respectively). Noteworthy, isthe fact that the initial concentration of DMSO during the covalentcondensing reaction between the carrier protein and the TFCS reagentshould reach a final 1:10-20 dilution (v:v), in order to prevent theformation of protein precipitates in the mixture. The condensationreaction occurring between the tetanus toxoid and TFCS should take placeat room temperature for 60-90 minutes, in order to achieve the completesynthesis of the tetanus toxoid-TFCS product. This intermediatederivative product still preserves a reactive amino group protectedchemically by a trifluoroacetyl group (see FIG. 9B), which issubsequently hydrolyzed after exposing the product to an additional 2-3hour incubation period at room temperature in a PBS solution, pH 8-8.5.The pH of this latter phosphate-buffered solution should be adjustedwith a solution of 10 N NaOH. Under these experimental conditions, thefree reactive amino groups in the conjugated TFCS compound are generatedat the deprotected end site of the tetanus toxoid-TFCS complex (see FIG.9B). This final tetanus toxoid-TFCS intermediate derivative product issubsequently exposed to purification procedures using standard dialysisprotocols. Briefly this method consists in incubating the tetanustoxoid-TFCS conjugate, placed inside a 10 kDa cut-off dialysis membrane(Sigma-Aldrich)] against three changes of 6 liters of 0.1 M phosphatebuffer solution, pH 7.2, at 4° C. every 8 hours during a 24 hour period.

7. Tetanus Toxoid-TFCS Intermediate Derivatives: Synthesis of the FinalStructural Formulation of the Bivalent Vaccine Against Morphine-HeroinAddiction

The covalent condensation of the intermediate product of morphine, theEDC-(M-6-H) to the tetanus toxoid-TFCS complex (see FIG. 10A) isachieved through a stable amide covalent bond, formed after the reactionbetween the free carboxyl groups exposed at the end of the EDC-(M-6-H)conjugate and the unprotected free amino groups from the TFCS reagentlinked to the tetanus toxoid (the TFCS-tetanus toxoid molecular complex,see FIG. 10B.

Due to that the complete synthesis and purification procedures of thetetanus toxoid-TFCS derivative requires at most a 24 hour period, thesynthesis of the EDC-(M-6-H) conjugate should be carried out just afterthe completion of the reaction-purification steps of the tetanustoxoid-TFCS conjugate. This is mostly due to the fact that theEDC-(M-6-H) product is very unstable and easily to hydrolyze whenexposed to prolong storage (i.e., more than two hours) even attemperatures below 10° C. Other important issues that requireconsiderable attention for the optimization of the covalent condensationbetween the EDC-(M-6-H)-conjugate and the tetanus toxoid-TFCS product,are referred to the concentration of the reactants added in the chemicalreaction. The tetanus toxoid used as the carrier protein, exhibits amolecular weight of about 100 kDa, containing an approximate number of68 lysine residues. The exposed ε-amino groups (active sites) in thelateral chain of these amino acid residues, allow the covalentcondensation of TFCS during the synthesis of the present invention ofbivalent vaccine against morphine-heroin. In this context, each μmol oftetanus toxoid (100 mg) contain an approximated density of up to 0.07μmol of available active sites (unprotected free amino groups) thatcould be covalently linked with the TFCS reagent. Furthermore, ourcondensation reaction between the EDC-(M-6-H) intermediate derivativeand the tetanus toxoid-TFCS conjugate uses a stoichiometric mol:molratio of 100 μmol of the EDC-(M-6-H) for each 0.07 μmol of amino groupsof the tetanus toxoid-TFCS conjugate.

In a typical formulation of the covalent condensation between theEDC-(M-6-H) intermediate derivative and the tetanus toxoid-TFCSconjugate, the concentration of reagents and the reaction conditionsrequire the mixture of 100 mg of tetanus toxoid-TFCS complex (1 μmoltetanus toxoid-TFCS=0.07 μmol of free amino groups/active sites) plus 30mg of the EDC-(M-6-H) intermediate derivative (70 μmol of active sitesfor covalent condensation), dissolved in 100 ml of 0.1 M phosphate/0.15M NaCl buffer solution, adjusting the pH to 7-7.5 (being the calculatedmolecular mass of the latter EDC-(M-6-H) compound of up to 426.37daltons). The reacting mixture is incubated at room temperature during2-3 hours under constant stirring. The synthetic product obtained, themorphine-6-hemisuccinyl-tetanus toxoid vaccine, is then purified usingstandard dialysis procedures using 10 kDa cut-off dialysis membranes(Sigma-Aldrich) against six changes of 6 liters of 0.1 M phosphatebuffer solution, pH 7.2, at 4° C., over a 48 hour period, in ordereliminate the by-products formed during the reaction, such as urea andthe non-haptenized EDC-(M-6-H) intermediate derivative.

Once the purification of the morphine-6-hemisuccinyl-tetanus toxoidvaccine has been completed, the dialyzated solution is subsequentlysterilized by filtration in 0.45 μm pore size membrane filters (GelmanSci) under positive pressure. Finally, 1 ml aliquots of the filteredsolution are dry-frozen, lyophilized in sterile glass vials, sealedunder vacuum and preserved under storage at 4° C. Several agents usednormally to stabilize and prevent degradation of conjugates duringdry-freezing and storage procedures (E. Harlow and D. Lane, Antibodies;A Laboratory Manual, Cold Spring Harbor Laboratory, New York, (1988) canbe added to the therapeutic formulation of the present invention of thebivalent vaccine against morphine-heroin addiction. Examples of selectedagents, consist of jelly, peptone, dextrine, methyl-cellulose, sucrose,lactose, maltose, glucose, fructose, sorbitol, glycerol, manitol,inositol, citric acid, tartaric acid, polyethylenglycol, andpolyvinylpirrolidone, among many others. Each vial of the bivalentvaccine product against morphine-heroin addiction contains an averagedose of about 1 mg of dry-frozen product of tetanus toxoid used as“reference dose unit”. The protein concentration of each dose unit ofthe bivalent vaccine was determined by a standard protein quantificationmethod using a bicinchoninic acid reaction kit, according to theprocedures recommended by the manufacturer (Pierce Chemical). Thequantitative measurement of percentage of incorporation of theEDC-(M-6-H) intermediate derivative covalently condensed to the freeamino groups of the tetanus toxoid-TFCS complex was carried out bystandard titration procedures, using the o-phtaldehyde reagent fortittering the number of free amino groups of the tetanus toxoid-TFCSintermediate derivative (J. Cashman et al, J. Pharmacol. Exper. Ther.293: 952-961, 2000). Percentage yield of up to 75-85% of haptenconjugation (morphine) with the carrier protein (tetanus toxoid) arenormally achieved in the present formulation of the bivalent vaccineagainst morphine-heroin addiction.

The carrier protein used in this bivalent vaccine can be selected amongmany other proteins such as ovalbumin, rabbit serum albumin,thyroglobulin, fibrinogen, KLH, goat erythrocyte membranes and flagellinas well as toxoids from diphtheria, cholera and botulinic toxins, whichmay be covalently linked to the M-6-H intermediate derivative, using thesynthetic conjugation procedure with the EDC and TFCS described above.The final product obtained may be used then, in active immunizationexperiments against morphine-heroin and/or used as solid-phase adsorbedantigens in immunological assays (i.e., ELISAs).

In the present invention, a morphine-6-hemosuccinyl-BSA conjugate wassynthesized in parallel to the present invention of the bivalent vaccineagainst morphine-heroin addiction using similar synthetic protocols forthis latter vaccine. The rationale to synthesize this additionalmorphine conjugate, was for using it as a morphine antigen adsorbed tothe solid phase of our antibody-capture ELISA immunoenzymatic assays.These latter assays were used to identify, monitor, quantify (FIGS. 1-4)and validate the specificity (FIG. 5) of the humoral immune responseinduced by active vaccination with the therapeutic formulation of thepresent bivalent vaccine against morphine-heroin addiction.

8. Molecular Structure of the Bivalent Vaccine Against Morphine-HeroinAddiction

The structural formulation of the present invent of the bivalent vaccineagainst morphine-heroin addiction shows for the first time the use ofthe chemical reagent, TFCS, used for the synthesis of a longspacer-linker arm to haptenize morphine and/or heroin to the tetanustoxoid.

A total molecular size of up 20.15 Å is calculated for the spacer-linkerarm that separates the haptenized morphine covalently linked through the6-carbon atom in its phenantrenic ring structure. The size of thisspacer linker arm is significantly longer (see FIG. 11) from those usedto synthesize previous reported morphine immunogens. This latterimmunogens used the EDC reagent as homobifunctional cross-linker tocovalently link the 3-O-carboxymethylmorphine and/or M-6-H to theε-amino groups of exposed lysine residues in either BSA or KLHmolecules, used as carrier proteins. For instance, the immunogenicconjugate of morphine-6-hemisuccinyl-BSA or morphine-6-hemisuccinyl-KLHcontain a spacer-linker arm size of about 12.4 Å, because they lack the6 carbon atom extension produced by the hydrocarbonated chain of theTFCS reagent. The addition of this hydrocarbonated chain from the TFCSin our vaccine formulation increases the length of the total spacerlinker arm by about 7.74 Å, (see FIG. 11). As mentioned above, thisstructural innovation of the increased length of the spacer-linker armin our novel model of anti-morphine-heroin vaccine model disclosed inthe present invention shows important functional capabilities. These aredemonstrated by the following experimental findings: a) highimmunogenicity generated against haptenized morphine and/or heroin; andb) a superior capacity to triggers a robust humoral immune response withhigh and sustained titers of specific serum anti-morphine antibodies(FIGS. 1-4) which display equivalent cross-recognition to this opiateand its structural analogue, heroin (FIG. 5). Moreover, it is feasibleto hypothesize that the increased length of this new spacer-linker armintroduced in the structural formulation of our bivalent-vaccine modelagainst morphine-heroin addiction, offers structural and functionaladvantages, based on the humoral immune response produced by thisimmunogen, where sera antibodies cross-recognize with equivalentspecificity the immunogenic epitopes exposed by the haptenized morphinemolecule to the immune system of vaccinated animals which are shared byheroin and their endogenous metabolites 3-monoacetyl-morphine and the 3-and 6-morphine-glucuronides (FIG. 5). Additionally, the activevaccination with our novel morphine-heroin bivalent immunogen, disclosedin the present invention, may be used as an effective therapeuticprocedure to induce a robust humoral immune response able toimmunoprotect against the acquisition of addictive behaviors to thesetwo opiate compounds in the actively vaccinated host. Finally, thishumoral immune response induced by active vaccination in the host, mayoffer an immunoprotection against the endogenous activity of theaforementioned endogenous metabolites of both morphine and heroin, shownto display reinforcing addictive properties in detoxified and abstinentsubjects from their addiction to these opiate compounds (FIGS. 6 and 7).

In short, the spacer-linker arm exhibits a molecular size of 20.15 Å,where the 7.74 Å middle segment corresponds to the hydrocarbonatedbackbone introduced by the TFCS reagent, which has been covalentlyconjugated to the ε-amino groups of exposed lysine residues in thetetanus toxoid carrier protein; the 7.44 Å end-segment comprises theα-carbon atom and the next four carbon atoms of the lateral chain oflysine residues and the 4.97 Å condensed segment comprise thehemisuccinyl residue, which has been covalently linked via an estergroup to the 6-carbon atom of the phenantrenic ring structure of themorphine molecule, as shown in FIG. 11.

In addition to the synthetic, structural formulation, purificationprocedures, and therapeutic uses of the disclosed invention of thebivalent vaccine against morphine-heroin addiction, it is also revealeda complementary synthetic and purification procedures of anotherstructural formulation of this bivalent vaccine against morphine-heroinaddiction. This additional structural formulation of ananti-morphine-heroin vaccine consists in the alternate synthesis of anEDC-3-O-carboxymethylmorphine derivative product, using same syntheticprotocols and procedures previously reported in the literature (S.Spector and C. W. Parker, Science, 168:1347, 1970; S. J. Spector, J.Pharmacol. Exp. Ther, 178:253, 1971; H. Van Vunakis et al., J.Pharmacol. Exp. Ther, 180:514, 1972 and S. Gross et al., Immunochemistry11:453-456, 1974). This EDC-3-O-carboxymethylmorphine derivative wasalso covalently linked to the tetanus toxoid-TFCS conjugate according tothe synthetic procedures used to synthesize the structural formulationof the bivalent anti-morphine-heroin vaccine in the present invention,using the synthetic procedure as shown in FIG. 12.

This alternate model of the bivalent anti-morphine-heroin vaccinedisplays a different spacer-linker arm structure with a total molecularsize of 16.47 Å, where the 9.03 Å right-hand segment comprise thehydrocarbonated backbone introduced by the TFCS reagent, linked throughan amide covalent bond to the EDC-3-O-carboxymethyl residue in thephenantrenic ring structure of the morphine molecule. The 7.44 Åleft-hand segment comprise the α-carbon atom and the four carbon atomsof the lateral chain of lysine residues of the tetanus toxoid, whichhave been covalently linked through the ε-amino group to the left-handside end-segment of TFCS reagent, as depicted in the formula as shown inFIG. 13.

Adjuvants

Despite of high molecular mass and the multiplicity of immunogenicepitopes displayed by the immunoconjugates containing covalently linkedhaptens of low structural complexity, as shown by our novel vaccinemodel against morphine and heroin addiction, its administration to asubject requires the supplement of adjuvant compounds, known to strengththe initial immune response (E. Harlow and D. Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York, pp 96-97,1988). In this context, adjuvants have the capability to induce a potenthumoral and/or cellular immune response to large types of antigens whichincludes, carbohydrates, peptides and proteins. Therefore, severalchemical formulations of adjuvants have been used and validated inactive vaccination protocols in animal species, which includecommercially available formulations, such as water-oil emulsions thatmay or may not contain Mycobacterium tuberculosum inactivated by heatexposure (Sigma-Aldrich), RIBI (RIBI Immunochem Research, Inc.) besidesother formulations containing biodegradable polymers and liposomes (seereview in J. Kohn et al., J. Immunol. Methods, vol. 95, pp 31-38, 1986).

After extensive decades of experimental research, the very fewauthorized and approved adjuvants used for human vaccination comprisedformulations containing aluminum hydroxide. The preparation of apharmaceutical composition or therapeutic formulation that includes the“bivalent vaccine against morphine and heroin addiction” in the presentinvention can be carried out using standard techniques handled by fieldexperts, together with any of the accepted vehicles, auxiliaries and/orpharmaceutical excipients described in the art of the technique,including, with no limitation, different adjuvant substances. A typicaldosification formulation of the bivalent vaccine against morphine-heroinaddiction and adjuvant used for active vaccination protocols in bothanimal and humans, consist in the preparation of a mixed ratio of 1:2(v:v) of the bivalent vaccine:aluminum hydroxide by mixing 1 ml of thevaccine resuspended in sterile deionized H₂O; with 2 ml of a stocksolution of 45 mg/ml of aluminum hydroxide (Imject-R-Alum, Pierce) addedby slow dripping (in no less than 3 minutes). The mixture of thereactants are incubated under slow and constant stirring for 1-2 hoursat room temperature. The final concentration of aluminum hydroxideshould not exceed 1.12-2.25 mg/100 μl in the reaction during the mixingprocess with the bivalent vaccine against morphine-heroin addiction.After the mixture has been completely stirred, the formulation of thebivalent anti-morphine-heroin vaccine/aluminum hydroxide adjuvant shouldbe loaded into sterile plastic syringes, using the parenteral route(i.e., subcutaneous, intramuscular and intraperitoneal) as preferentialadministration routes to introduce the vaccine formulation into thehost, with the exception of the intravenous route.

Other available immunogenic adjuvants that can be combined andadministered with the present invention of the bivalent vaccine againstmorphine-heroin addiction includes a large group of compounds, such asaluminum phosphate, interferons, interleukins, polylactic acid esters,biodegradable copolymers consisting in polyglycolic acid esters,liposomes, bacterial membranes lipopolysaccharides, bacterialmuropeptides and RIBI. These formulations and/or compositions adopt thepharmaceutical forms of injected solutions, suspensions, powders andsimilars compounds.

Active Immunization

The intramuscular route is the preferred parenteral route by means ofwhich the present invention of the bivalent anti-morphine-heroin vaccinemixed with aluminum hydroxide adjuvant should be administered tosubjects, although, other parentental routes, such as the subcutaneousand intraperitoneal, may be used for vaccination protocols. The presentinvention of bivalent vaccine against morphine-heroin addiction, or thepharmaceutical composition or therapeutical formulation containing thisimmunogenic vaccine preparation, should be administered using atherapeutically effective dose and a established dose-administrationprotocol/regimen just only in the abstinent and detoxified subject fromtheir previous morphine and/or heroin addictive behavior. This protocolshould always be adjusted according to the degree of both complaint andaddiction of the individual. A typical active immunization schedule usesthe intramuscular route to inoculate this vaccine formulation in adose-unit of the haptenic drug-carrier protein conjugate of up to 1-2mg/kg of the individual's body weight (i.e., male rats of 250-350 mgweight, Wistar or Sprague-Dawley strain). This priming inoculation mustbe subsequently followed by 3-6 reboosting periods, administered at14-day intervals, by administering the same dose-unit of this vaccineformulation during reboosting. The active immunization in controlsubjects is carried out only with adjuvant (aluminum hydroxide) or withadjuvant plus carrier protein (aluminum hydroxide+tetanus toxoid). Theserum obtained from vaccinated subjects should be sampled 10-12 daysafter each reboost using standard protocols and procedures previouslyreported (E. Harlow and D. Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York, 1988) to monitor the humoral immuneresponse against morphine and heroin, including to its endogenousmetabolites. To achieve these experimental conditions, differentvaccinated experimental animals were bled (100 μl/animal) and the serafractions were obtained after collected samples were subjected to bloodclotting for 24 hours at 4° C., followed by centrifugation of theclot/supernatant fraction at 14,000×g. The obtained serum fractions wereimmediately frozen at 20° C. until use.

Antibody capture ELISA immunoenzymatic assays were used to identify andmonitor the humoral immune response against both opiate substances,after each reboost, according to the active immunization proceduredescribed above. These results allowed to support the efficacy of ournovel anti-morphine-heroin vaccine to induce a robust humoral immuneresponse against these opiate compounds. Moreover, these results led toidentification of the number of reboosts required to induce a humoralresponse with maximum and stable levels of serum antibodies againstthese opiate substances. Altogether, these experimental data were usedto select and define candidate hyperimmune animals that weresubsequently exposed to the immunoprotection protocol against theseopiate substances using the rat behavioral model of the addictiveintravenous opiate self-administration paradigm (see below FIGS. 6 and7).

A typical immunoenzymatic procedure of antibody capture ELISA assay usedto monitor the humoral immune response against morphine and heroin fromthe serum of actively vaccinated subjects with our therapeuticantimorphine-heroin vaccine formulation consists in the initialsynthesis of the solid phase of the assay by enhancing the adsorption of3-4 μg of the antigenic preparation of morphine-6-hemisuccinyl-BSA/wellin 96-well plates (Immunolon I, Corning). The captured ofanti-morphine/anti-heroin antibodies by the antigenic fraction absorbedonto the solid phase is carried out after a 6 h incubation period atroom temperature of aliquots (50 μl/well) containing progressive serialdilution of antibodies obtained from immunized animals (i.e., 1:10,1:100, 1:1,000, 1:10,000 and 1:1,000,000). Thereafter, the wells areextensively washed with a solution containing 1% BSA/0.3% Tween-20/PBS,pH 7.4, followed by 2-3 hours incubation period at room temperature witha secondary anti-IgG (H+L) rat antibody (Vector Laboratories) conjugatedwith horseradish peroxidase. After this incubation period, the wells areextensively washed to remove the excess of the unbound secondaryantibody, followed by the detection of immunopositive signals/well usinga chromogenic substrate (OPD, SIGMA). The assayed wells are exposed tospectrometric detection of the absorbance values at 490 nm of theantibody fraction captured by the antigenic solid phase using amicroplate ELISA-detector system. The obtained spectrometric absorbancevalues reflect the amount of antibody captured by the antigenic solidphase. Thus, the final antibody titer values are estimated andexpre3ssed as the inverse value of the diluted fraction of antiseratested that gives 50% of the maximum absorbance response, using computerstandardization procedures.

FIG. 1 shows a representative result of an antibody capture ELISA assayused to identify initially the efficacy of our novel bivalent vaccineagainst morphine-heroin addiction to induce a humoral immune responsewith high antibody titers (i.e., producing an average titer value of≈1:100 000) against morphine, shortly after the second reboost in agroup 10 sampled immunized animals. As shown in the figure, theconcentration of reactive antibodies detected through its absorbance at490 nm in the assay decreases proportionally to the serial dilution ofthe antisera.

FIG. 2 depicts a representative result of an antibody capture ELISAassay for monitoring the time-course of serum titers of antibodies formorphine-heroin after reboosting animals (1-7 reboosts) periodicallywith the bivalent vaccine preparation against morphine-heroin addiction.After priming rats (first inoculation) with this novel therapeuticformulation of the bivalent vaccine against morphine-heroin addiction,the serum antibody titers against these opiate substances were monitored10-12 days after each reboost (from 4-7). As shown in the figure, aprogressive increase in antibody concentration against these opiatesubstances was obtained up to the fourth reimmunization period, where 10actively vaccinated animals exhibited mean titer values ranging from1:800,000-1:1000,000. However, the subsequent reimmunizations with themorphine-heroin immunogen (from the 5-7th) were not effective ininducing significant increasing antibody titers in animals consideredhyperimmune to these opiate drugs (data no shown in the figure). Thislatter result postulates the use of short-term active immunizationprotocols with our novel therapeutic anti-morphine-heroin vaccineformulation to reach a maximum humoral immune response against bothopiate substances.

One of the central goals to be reach by every novel model of therapeuticvaccines when used in active immunization protocols, is their capabilityto induce a robust and stable over time humoral and/or cellular immuneresponse established with long-term immune memory. In this context, theFIG. 3 is a plot of representative data showing a temporary decrease ofthe humoral immune response of antibodies against morphine-heroin seenafter the fourth reimmunization in actively vaccinated subjects (n=10)with our novel therapeutic vaccine formulation. Noteworthy is the factthat non-rebbosted hyperimmune animals, show a progressive time-coursedecrease of antibody titers along a 120 day period. The initial antibodytiter obtained after the last re-immunization (which averaged between1:800,000-1:1000,000) showed a significant decreased of about 40-50times, reaching an average minimum titer value of 1:20,000 at the end ofthis period of time. These data show and support the hypothesis thatactive immunization with our novel therapeutic anti-morphine-heroinvaccine formulation is able to induce a classical humoral immuneresponse reaching stable antibody titers, at least after the fourthre-immunization. This evidence is strongly supported by the experimentaldata obtained from antibody capture ELISA as depicted in FIG. 4, whichshows that a long-term immune memory response against these two opiatesubstances has been established after the fourth reboost. This figureshows the average values of serum antibody titers of ten experimentalsubjects exposed to a subsequent reboost with the bivalent vaccine aftercompleting a long-term non-reboosting period of six months from the lastre-immunization (4^(th)). As shown in the figure, active re-immunizationwith the present invention of the therapeutic anti-morphine-heroinvaccine formulation induced a rapid and stable recovery of thepre-existing maximum levels of antibody titers against these opiatesubstances (i.e. usually within the first 5-10 days after reboosting) innon re-immunized hyperimmune animals. It is worth to note the similartime-course decrement of antibody titers shown after the fourth reboost(see FIG. 3) in non-vaccinated hyperimmune animals (i.e., afterchallenging animals with the latter vaccine reboost, the maximum titerlevels were reached 15-20 days after reboosting, followed a slow andprogressive linear decreased during the next 30 days, reaching thelowest levels of detected antibody titers up to 120 days, data not shownin figure).

Once the efficacy of our novel therapeutic formulation of ananti-morphine-heroin vaccine was evaluated and validated in activevaccination protocols by showing its capability to generate a robusthumoral immune response characterized by high and sustained serumantibodies titers against these opiate substances, additionalimmunoenzymatic competitive ELISA assays were designed and developed toevaluate and identify the specificity of the anti-morphine-heroinantibodies.

This competitive ELISA assay used to evaluate the antibody specificityis based on the same experimental design used for the aforementionednon-competitive ELISA assays. The difference to the competitive ELISAassays consists in a preadsorption step of the specific antisera fromhyperimmune animals using different concentration (i.e. that range inthe nM-μM range) of potential competitive antigens potentiallycross-recognized by the anti-morphine antibodies. These competitiveantigens included morphine as the positive control substance in additionto the three main endogenous metabolites of morphine and heroin (e.g.,6-monoacetylmorphine, morphine-3-glucuronide and morphine-6-glucuronide)shown to display opiate-reinforcing properties, and heroin, as thesynthetic structural analogue of morphine, shown to exhibit at least aan order of magnitude higher in its opiate-reinforcing properties atequivalent dose, than its natural opiate ortholog, morphine. Two otherrepresentative endogenous opioid peptides produced in the CNS ofmammals, such as leucine-enkephalin and β-endorphin, were also includedas competitive antigens in this assay. Furthermore, this assay alsoincluded pharmacological active competitive antagonists compounds foropioid receptors, such as naltrexone, a commonly used substance in themaintenance of abstinence from heroin addiction in the humans.

As this assay is based in the detection of positive signals originatedfrom the absorbance emitted from the reacting wells when exposed to themicroplate ELISA detector system at 490 nm, wells exhibiting an absenceof significant signals at this wavelength (490 nm) suggest the lack ofspecific antibodies captured by the solid phase adsorbed antigen (whichin our case was the morphine-6-hemisuccinyl-BSA conjugate). Ifoccurring, this latter experimental condition would indicate that serumantibodies generated by active vaccination with our novelanti-morphine-heroin vaccine formulation would display potentialcross-recognition for some of the competitive antigens used in theassay.

The representative data depicted in FIG. 5 illustrate a competitiveimmunoenzymatic ELISA assay, which shows the equivalent specificity ofthe serum antibodies to cross-recognize morphine and heroin (note thatboth competition curves display similar competitive morphine and heroindoses in the range of up to 0.6-0.8 μM at the IC₅₀ reference values).Additionally, these assays also show the capability of suchanti-morphine-heroin serum antibodies to cross-recognize differentbiotransformation metabolites from these opiate substances (i.e.,6-monoacetyl-morphine, morphine-3-glucuronide andmorphine-6-glucuronide). Furthermore, no cross-recognition to othersubstances such endogenous opiate peptides and the opiate receptorantagonist naltrexone was observed in same assays. Collectively, theseresults make feasible to propose the potential lack of immunologicalinterference of this immunogen in active vaccination protocols when itcould be used in humans treated with classical anti-addictive therapiesusing morphine structurally dissimilar opiate medications opioid such asnaltrexone, naloxone, methadone and buprenorphine. Moreover, it may beassumed that our new therapeutic vaccine formulation againstmorphine-heroin addiction is not able to generate an autoimmuneresponse, because the antibodies generated by this vaccine are not ableto cross-recognize endogenous opioid peptides (i.e., leucine-enkephalinand β-endorphin) that besides to be synthesized in the brain inhyperimmune vaccinated animals including humans, they do participate inthe regulation of a multiple array of physiological activities andprocessing of a wide range of brain functions in the CNS of mammals.

Evaluation and Validation of the Efficacy of the Present Invention ofthe Therapeutic Bivalent Vaccine Formulation Against Morphine-HeroinAddiction

After showing the validation of the efficacy of the present therapeuticbivalent anti-morphine-heroin vaccine formulation to conferhyperimmunicity against morphine and heroin with an enhanced long-termimmune memory response, through the generation of high and sustainedserum titers of specific reacting antibodies against these opiate drugsand their endogenous metabolites in immunized subjects, we decided toexplore the immunoprotective effects of the present therapeutic vaccineformulation against the re-acquisition of addictive intake behavior inhyperimmune animals detoxified and abstinent from addiction to theseopiates. In this context, tested hyperimmune against morphine/heroinanimals were exposed to operant behavioral tests using the intravenousdrug self-administration paradigm for both morphine and heroin. Thesepharmacological paradigms used in the animal model of the rat wereimplemented from related pharmacological paradigms previously reportedby several research groups (J. M. Van Ree et al., J. Pharm. Exp. Ther.,204 (3): 547-557, 1978; J. M. Van Ree and D. de Wied, Life Sci.21:315-320, 1977; T. J. Martin et al., J. Pharmacol. Exp. Ther.272:1135-1140, 1995; P. Hyytia et al., Psychopharmacology, 125:248-254,1996; T. J. Martin et al., Brain Res. 755:313-318, 1997; C. W. Hutto,Jr. and W. F. Crowder, Pharmacol. Biochem. Behay. 58(1):133-140, 1997;R. Ranaldi and E. Munn, Neuroreport, 9:2463-2466, 1998; S. Martin etal., Brain Res. 821:350-355, 1999; I. M. Maisonneuve and S. D. Glick,Eur. J. Pharmacol, 383:15-21. 1999; S. D. corner et al.,Psychopharmacology, 143327-338, 1999; S. Semenova et al., Eur. J.Pharmacol. 378:1-8, 1999; M. R. A. Carrera et al., Psychopharmacology,144:111-120, 1999; Z-X. Xi and E. A. Stein, J. Pharm. Exp. Ther.290:1369-1374, 1999 and L. J. Sim-Selley et al., J. Neurosci.20(12):4555-4562, 2000).

The pharmacological models of intravenous self-administration paradigmsof both morphine and heroin in the rodent have been widely used toexplore the neurobiological mechanisms by which these opiate producedtheir drug-reinforcing properties. Additionally, these models have beenalso used to evaluate the anti-addictive effects of therapeuticcompounds such as methadone, naloxone and naltrexone. Moreover, thesepharmacological paradigms are extremely useful to evaluate themotivational and drug-reinforcing responses, independently from thedirect pharmacological effects produced by these opiate drugs in thenervous system (i.e, psychomotor activation) when proper protocols areemployed during the pharmacological self-administration of thesesubstances. Therefore, in order to evaluate and validate theimmunoprotective effects against morphine-heroin addiction conferred bythe present invention of the therapeutic bivalent anti-morphine-heroinvaccine formulation, our laboratory designed, developed and validated anintravenous drug self-administration paradigm for these two opiatesubstances in the animal model of the rat.

a). Development, Implementation and Validation of the IntravenousSelf-Administration Paradigm of Morphine and Heroin in the Animal Modelof the Rat

The pharmacological model of the intravenous morphine/heroinself-administration paradigm in the rat was standardized from severalprotocols previously reported by different groups (J. M. Van Ree et al,J. Pharm Exp. Ther., 204 (3):547-557, 1978; J. M. Van Ree and D. ofWied, Life Sci. 21:315-320, 1977; T. J. Martin et al., J. Pharmacol.Exp. Ther. 272:1135-1140, 1995; P. Hyytia et al., Psychopharmacology,125:248-254, 1996; T. J. Martin et al., Brain Res. 755:313-318, 1997; C.W. Hutto, Jr. and W. F. Crowder, Pharmacol. Biochem. Behay.58(1):133-140, 1997; R. Ranaldi and E. Munn, Neuroreport, 9:2463-2466,1998; S. Martin et al., Brain Res. 821:350-355, 1999; I. M. Maisonneuveand S. D. Glick, Eur. J. Pharmacol, 383:15-21. 1999; S. D. Comer et al.,Psychopharmacology, 143327-338, 1999; S. Semenova et al., Eur. J.Pharmacol. 378:1-8, 1999; M. R. A. Carrera et al, Psychopharmacology,144:111-120, 1999; Z-X. Xi and E. A. Stein, J. Pharm. Exp. Ther.290:1369-1374, 1999 and L. J. Sim-Selley et al., J. Neurosci.20(12):4555-4562, 2000). Basically, this pharmacological model consistsin using surgically implanted animals with teflon sterile cathetersplaced into the right or left external jugular vein to opiateintravenous self-administration paradigms using morphine and heroin asdrug-reinforcers, during 4 h/daily sessions, inside operant conditioningSkinner boxes, controlled by the observer using computerized signals. Inthis context, the intravenous infusion of a complete “dose-unit” of eachof these two opiate substances is established by the fixed number ofoperant lever responses made by the animal on a retractile lever (placedon the front panel of Skinner boxes) at specified time intervals. Forexample, the infusion of a dose-unit of morphine (i.e. 1900 μg/0.2 ml/kgof weight) and heroin (60 μg/0.2 ml/kg) are carried out when the animalcompletes a fixed number of lever responses (i.e. 1, 3, 5, 10) after adefined time intervals (i.e., 20, 40, 80 seconds), time at which theretractile lever is inactive. Thus, under this pharmacologicalconditions, one can evaluate the drug-intake behavior responses, byestimating in the 4 hour/daily sessions the total number of opiateinfusions made by the animal. Also included in the analyses are themeasurements of drug-seeking behavior responses by estimating the totalnumber of lever retractions occurring at the time-intervals, when theretractile lever is inabilitated. Under these experimental conditions,trained animals established the amount of opiate drug require to beself-administered. Thus far, this pharmacological paradigm allows tocarry out quantitative and reproducible procedures used to estimate theaccumulated doses of intravenously self-administereddrug/animal/session/day, including the accumulated doses ofself-administered drug/animal throughout the training schedule (i.e.,accumulated data over 15, 30, 60 days). The capability of morphine andheroin to induce an operant behavioral response (i.e, manipulation ofthe retractile lever to produce a and/or drug-seeking behaviors) isdefined as the reinforcing properties of each drug to discriminate thedrug-associated stimulus. In this context, hiperimmune vaccinatedanimals with the bivalent vaccine of the present invention, with immunehumoral responses of high and sustained anti-morphine-heroin serumantibody titers, should blunt or neutralize the drug-reinforcingproperties induced by these opiate substances in the brain, whenchallenged to acquire the addictive intravenous self-administeringbehavior of either morphine or heroin. These animals should show asignificant decrease of opiate drug-taking and drug-seeking behavioralresponses due to the absence of reinforcing drug-associated stimuli.

In summary, this pharmacological model based on the intravenous opiateself-administration paradigm, allowed us to obtain and constructbaselines of the operant drug-intake behavior in animals thatconsolidated addictive responses to both morphine and heroin. Thepharmacological parameter concerning the opiate-intake behavioralresponses to both morphine and heroin were obtained after comparing theself-infusion rates of these drugs in hyperimmune animals immunized withthe therapeutic bivalent anti-morphine-heroin vaccine formulation of thepresent invention and control groups (non-immunized or immunized onlywith adjuvant and adjuvant plus carrier protein, see representativeresults in FIGS. 6 and 7).

1. Functional Development and Implementation of Skinner's Operant Boxes.

The installation and functioning of eight Skinner's operant boxes(aluminum and transparent acrylic) designed for intravenousself-administration of liquids and drugs in the rat animal model weredeveloped according to the operating standards recommended by themanufacturer (Operant Behavior Conditioning Systems for lab animals, TSESystems, Hamburg, Germany).

2. Development of Conditioning Learning Training Paradigms for LeverPress and Food Reward.

Wistar male rats (260-320 g) were trained to localize and pressretractile levers within the operant Skinner boxes, and for each leverpress, animals were rewarded with a maximum of 200 food pellets (45 mg)(Noyes Traditional Food Precision Pellets; Research Diets, Inc.,Lancaster, N.H.) during 5-7 days in a 4 h training sessions. Under theseexperimental conditions, animals were conditioned to obtainedfood-reward (reinforcing stimulus) each time they pressed the retractilelever [fixed reinforcement protocol 1(FR1)], upon exposure of a cuelight stimulus (conditioned stimulus), controlled online by software(TSE, OBS system) during daily 4 h sessions for a period of 5-7 days.After this training period, the duration of the sessions were shortenedto 30 minutes, increasing the time-out intervals from 5 (TO-5) to 20 sec(TO-20), time at which retractile levers were inabilitated during thenext following 3-5 days. Thus, animals were trained to complete theirlever responses by obtaining only 50 pellets under a fixed reinforcementschedule (FR1, T0-20 sec) in a daily 30-minute sessions. Animalssucceeding in this behavioral conditioning training, were returned totheir individual home cages, under restrict diet (16-20 gr foodpellets/day), and subsequently exposed to the surgical implantation ofTeflon intravenous catheters into the external jugular vein, so as toinitiate the experimental procedures of immunoprotection againstmorphine/heroin addiction when exposed to the intravenous opiateself-administration paradigms.

3. Surgical Implantation of Sterile Catheters into the External JugularVeins.

Experimental animals trained for lever press and food reward, using theoperant conditioning behavior described above, were subjected to generalanesthesia and surgical aseptic conditions for the surgical implantationof teflon sterile catheters within the right or left external jugularveins. The whole surgical procedure was performed according to standardsurgical protocols described by K. M. Kantak et al. (Psychopharmacology,148:251-262, 2000). After surgery, animals were returned to theirhome-cages and the functional viability of the implanted catheters werechecked in a daily basis by infusing saline solution and antibiotics [5%Enrofloxacyn (0.50 mg/kg); Gentamicyn-Super 5 mg/kg). After seven daysof post-surgical recovery, animals were then subjected to thepharmacological paradigms of intravenous self-administration of bothmorphine and heroin.

4. Development and Establishment of Baseline Responses of IntravenouslySelf-Administered Morphine and Heroin.

The functional viability of implanted catheters in post-surgicallyrecovered animals was verified prior to exposing animals to 4 hour/dailysessions of our intravenous morphine and heroin self-administrationparadigm. Initially, separate groups of animals were exposed to thecontingent self-administration of a fixed dose-unit of morphine (1900μg/kg/0.2 ml saline) during 10 seconds injection) or heroin (60μg/kg/0.2 ml of saline/10 seconds injection) following a fixedreinforcement schedule (FR1) TO-20 seconds, during 4 hour-daily sessionsfor 5-7 consecutive days. The difference of these reinforcing dose-unitvalues between morphine and heroin was based on data previously reportedin the literature (J. M. Van Ree et al, J. Phar. Exp. Ther.204(3):547-557, 1977 and C. W. Hutto, Hr. and W. F. Crowder, Phar.Biochem. Behay. 58(1):133-140, 1997) which showed that a morphine:heroindose-ratio relationship of 32:1, produces equal choice on theself-infusion of these opiate substances when self-administered by therat under this experimental conditions. This training period led animalsto acquire stable baseline responses on the contingentself-administration of these opiates, over an additional training periodof 7-10 days. Under these protocol conditions, trained animals producedaverage baseline infusion-responses of 25±3 and 20±5 duringself-administration of the fixed dose-units of both heroin and morphine,respectively. Baseline self-infusion responses to these two drugs wereconsidered established and consolidated when the variability coefficientvalues varied no more than 10% for each drug along self-infusionsessions, for at least five consecutive experimental days. Once theinitial baseline self-infusion responses to both morphine and heroinwere achieved, the initial extinction phase was carried out bysubstituting the opiate substances for vehicle solution (i.e. vehiclesolution=saline 0.9% NaCl in sterile deionized H₂O) during the nextfollowing 3-5 days, just after baseline self-infusion responses to bothopiates were established. The extinction responses to self-infusion ofboth morphine and heroin responses achieved by surgically implantedanimals were defined after achieving a mean average number of extinctionresponses/session/day of 3±2 to the self-administered vehicle solution,in groups of animals trained to self-administered either morphine orheroin that consolidated an initial phase of baseline responses asmentioned above. To consolidate the opiate self-administration behaviorresponses to both morphine and heroin, two subsequentre-acquisition-extinction cycles of opiate self-administration wereperformed. In this experimental context, fifteen days after obtainingthe average baseline responses of the extinction phase to the opiateself-administration paradigm, we evaluated the antagonism effect of theanti-morphine-heroin serum antibodies on the re-acquisition of theself-infusion addictive behavior responses to both opiate substances inhyperimmune animals (trained to self-administer these opiate substances)after being actively immunized with the therapeutic bivalent vaccineformulation against morphine-heroin addiction, using thevaccination/immunization protocol disclosed in the present invention.

5. Characterization of the Immunoprotective Effect AgainstMorphine-Heroin Addiction Induced by Active Immunization with theAnti-Morphine-Heroin Vaccine of the Present Invention

Different groups of animals trained to self-administered morphine andheroin which established baseline self-infusions of these opiate drugswhere actively vaccinated with either the therapeutic morphine-heroinbivalent vaccine formulation of the present invention or controlcompounds (i.e., aluminum hydroxide used as co-adjuvant and thisco-adjuvant plus tetanus toxoid used as the carrier protein) followingthe same immunization protocol disclosed in the present invention. Oncethe humoral immune response against these two drugs (see FIGS. 1, 2, 3and 4) was established in hyperimmune vaccinated animals, they were thenre-exposed to the intravenous self-administration paradigm with bothmorphine and heroin, so as to assess the immunoprotective responsesagainst these opiate drugs by measuring the number of completeself-infusion responses (drug-intake behavior) throughout 15-20consecutive 4 hour-daily sessions. Same studies were carried out in thecontrol animal groups, which received either the adjuvant alone or theadjuvant plus the carrier protein.

Data obtained were expressed as the mean average of accumulated numberof complete self-infusion responses/day/in the experimental vaccinatedgroup during 15-20 daily sessions, and assayed to evaluate theimmunoprotective effect. The statistical analysis of data was performedby variance analysis (ANOVA), followed by a Newman-Keuls test forpost-hoc comparison analysis.

Under this experimental context, tested groups included, hyperimmuneanimals against morphine-heroin (CP-MORPHINE, n=8) and control groupsimmunized with either aluminum hydroxide adjuvant (ALUM, n=8) or withthe carrier protein plus adjuvant (CP+ALUM, n=8). All of them receivedsame dose-unit of heroin or morphine during the intravenousself-administration paradigm as disclosed previously in the presentinvention. The results showing the average baseline responses (values)of the number of self-infusions achieved for each self-administeredopiate substance, as well as the self-administered control vehicle(i.e., saline) along the 15-20 consecutive, 4 hour-daily sessions, areshown in FIGS. 6 and 7.

FIG. 6, depicts the immunoprotective effect induced by activevaccination with the therapeutic anti-morphine-heroin bivalent vaccineformulation of the present invention against the intravenous morphineself-administration behavior in the animal model of the rat. The groupof rats immunized with the vaccine of the present invention, and thecontrol groups, immunized with adjuvant or with adjuvant plus carrierprotein were exposed to the morphine self-administration paradigm.Control animals that received only aluminum hydroxide (ALUM) asimmunogen did not show significant changes with regard to the averageresponses of self-infusion of morphine/session (17±4, S.E.M.) comparedto the pre-immunization average responses in control animals (18±5,S.E.M.). Conversely, animals vaccinated with the immunogenic morphinepreparation (CP-morphine) showed a significant reduction in the averagenumber of heroin self-infusions/session (4±3, S.E.M., p<0.005) comparedto animals immunized with adjuvant (ALUM) or with adjuvant plus carrierprotein (CP-alone+ALUM). It is worth to note the similar pattern of themean average of self-infusion responses obtained with saline (controlvehicle) achieved by animals immunized with these three differentvaccine preparations (3±2 with ALUM, 3±2 with ALUM+CP; and 3±2 with theanti-morphine-heroin bivalent vaccine of the present invention).

FIG. 7, depicts the immunoprotective effect of the active vaccinationwith the therapeutic anti-morphine-heroin bivalent vaccine formulationof the present invention against the intravenous heroinself-administration behavior in the animal model of the rodent. Thegroup of rats immunized with the present vaccine and the control groupsimmunized with adjuvant and/or with adjuvant plus carrier protein wereexposed to the pharmacological paradigms of heroin self-administration.The control animals that received only aluminum hydroxide (ALUM) asimmunogen did not show significant differences with regard to the meanaverage of heroin self-infusion responses/session (24±4, S.E.M.) whencompared to the pre-immunization average responses (21±3, S.E.M.)obtained in control animals. Conversely, animals vaccinated with theimmunogenic preparation of morphine (CP-morphine) exhibited asignificant reduction in the average number of heroinself-infusions/session (6±2, S.E.M, p<0.005) compared to the animalsimmunized with adjuvant (ALUM) or with adjuvant plus the carrier protein(CP-alone+ALUM). Moreover, the average number of saline (controlvehicle) self-infusions reached by animals immunized with these threevaccine preparations (3±2 in the group of animals immunized with ALUM;2±3 in the group of animals immunized with ALUM+CP; and 3±1 in the groupof animals immunized with the anti-morphine-heroin bivalent vaccine ofthe present invention) were very similar.

Finally, the application of this kind of therapeutic strategies is beingevaluated for its future application in human subjects that exhibitserious addictive problems to both morphine and heroin.

Quite obvious to personal skilled in these techniques, that otheravailable variations, not specifically presented in the text above, maynevertheless be proposed within the scope of the present invention, andthus, they are included under the protection of this invent. Thus, thepresent invention is not just limited to the description of the specificmodalities presented as described above in the text, but clearlydepicted in the following patent claims.

The invention claimed is:
 1. A method of producing antibodies againstmorphine-heroin comprising administering to a subject a bivalentimmunogenic composition comprising a carrier protein (“CP”) and amorphinic product, wherein the CP and the morphinic product areconnected by a spacer-linker arm, wherein the immunogenic compositionhas one of the following structural formulas:

and wherein the immunogenic composition produces circulating polyclonalantibodies having equivalent cross-recognition to morphine and/or heroinin the blood, thereby preventing permeation of the composition into thebrain.
 2. The method of claim 1, wherein the immunogenic composition isadministered by subcutaneous administration.
 3. The method of claim 1,wherein the immunogenic composition is administered in an initialdose-unit of about 1-2 mg/kg according to the subject's body weight. 4.The method of claim 3, further comprising the steps of re-administeringthe immunogenic compositions 3-6 times at 14-21 day intervals.
 5. Themethod of claim 1, further comprising the step of isolating theantibodies.
 6. The method according to claim 1, wherein the antibodiesbind morphine/heroin metabolites.
 7. The method of claim 6, wherein themetabolites are selected from the group consisting of6-monoacetylmorphine, morphine-3-glucuronide, andmorphine-6-glucuronide.
 8. A method of producing antibodies againstmorphine-heroin comprising administering to a subject a bivalentimmunogenic composition comprising a carrier protein (“CP”) and amorphinic product, wherein the CP and the morphinic product areconnected by a spacer-linker arm, wherein the immunogenic compositionhas one of the following structural formulas:

wherein the antibodies bind morphine/heroin metabolites and wherein themetabolites are selected from the group consisting of6-monoacetylmorphine, morphine-3-glucuronide, andmorphine-6-glucuronide.